Amphoteric dissociation ion exchange medium and uses thereof and method for calibrating separation capacity thereof

ABSTRACT

An amphoteric dissociation ion exchange separation medium, the surface of which is an amphoteric dissociation covalently-modified layer. When an environmental pH value is lower than the isoelectric point, pIm, of the covalently-modified layer, the type of net charges on the surface of the covalently-modified layer is positive and the separation medium has the properties of an anion exchanger; when the environmental pH value is higher than the pIm, the type of net charges on the covalently-modified layer surface is negative and the separation medium has the properties of a cation exchanger. The separation medium has the properties of an anion exchanger and a cation exchanger at both sides of the pIm, respectively. The pH of an eluent can be adjusted to allow the separation medium surface and the target substance to have the same type of net charges, so that the target substance can be released by electrostatic repulsion.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Patent ApplicationNo. PCT/CN2018/111536, filed on Oct. 24, 2018, which claims the benefitof priority from Chinese Application No. 201711010305.0, filed on Oct.25, 2017. The contents of the aforementioned applications, including anyintervening amendments thereto, are incorporated herein by reference.

TECHNICAL FIELD

The application relates to an ion-exchange medium for separation ofbiologically active substances, and more particularly to an amphotericdissociation ion exchange medium and uses thereof and a method forcalibrating separation capacity thereof.

BACKGROUND

Ion exchange media are suitable for fast separation and analysis ofbiomolecules such as proteins and nucleic acids, purification andpreparation of proteins and nucleic acids, and chromatographicquantitative analysis of charged substances. These applications requirethat the ion exchange media have high adsorption capacity, high elutionefficacy to the adsorbed substances under mild conditions, accuratecalibration of separation capacity, low non-specific adsorption ofinterfering substances and high regeneration ability under mildconditions. Since classical ion exchange media cannot meet the aboverequirements simultaneously, the invention is intended to design a novelion exchange medium that meets the requirements as much as possible.

Under a given pH, surface groups of the separation medium dissociate andform ion pairs through electrostatic attraction with counter ions ofsmall size, and then the oppositely charged target substances areabsorbed based on electrostatic attraction and competitive binding toperform on-line chromatographic analysis of the target substances, orfurther, the adsorbed target substances are eluted by competitive ionsprovided by high concentration of monovalent neutral inorganic saltssuch as NaCl and KCl based on electrostatic attraction and competitivebinding. This is called ion exchange, and the separation media used areknown as ion exchangers.

The pH for ion exchange separation of biomolecules is usually between5.0 and 8.0, and is generally limited to between 3.0 and 11.0. When thetarget substance is purified or extracted by classical ion exchangers,both the adsorption and elution rely on properties of the targetsubstance, charged groups on the surface of the separation medium, andthe electrostatic attraction and competitive binding between ions in thesolution. There is usually only one type of ionizable groups on thesurface of the classical ion exchange separation medium, and there areonly two types of surface net charges at pH from 3.0 to 11.0. An ionexchange separation medium of which the surface net charge is positiveor zero but not negative is called an anion exchanger; and conversely,an ion exchange separation medium of which the surface net charge isnegative or zero but not positive is called a cation exchanger. Thereare still some defects in the performance of the classical ionexchangers for purification, preparation, extraction, concentration andchromatographic analysis of biomolecules. First, for goodhydrophilicity, the classical ion exchangers are provided with a largeamount of hydrogen bond-forming groups such as hydroxyl groups andamides, but such groups may form considerable hydrogen bonds with thetarget substance to generate non-specific adsorption, reducing theelution efficacy of the proteins/nucleic acids adsorbed by the classicalion exchangers, separation selectivity and capacity, and regenerationefficacy. Second, for a desirable elution efficacy, the monovalentneutral inorganic salt should be at a high concentration when used topromote the release of the target substance by competitive binding, andthe desalting is further required before the subsequentreanalysis/treatment, lowering the efficiency and increasing the cost.Therefore, there is a need to develop an ion exchange separation mediumbased on a new principle, which can not only significantly reducenon-specific adsorption but also improve the elution efficacy of theadsorbed target substance.

The classical ion exchangers cannot be applied to nucleic acidextraction, concentration and reanalysis due to low elution efficacy.Nucleic acids, as the genetic material, play crucial roles in biomedicalanalyses such as the current clinical diagnosis of infectious diseases,forensic evidence detection, genotyping, food safety detection, andconfirmation of sources of pharmaceutical raw materials. Nucleic acidshave low contents in biological samples and are usually detected afterPCR amplification. However, there are often a large number ofnon-nucleic acid substances in the samples that interfere with PCRamplification of nucleic acids. Therefore, rapid extraction of nucleicacids from the samples and removal of most interfering substances mustbe simultaneously performed before the PCR amplification. Nucleic acidshave phosphodiester bonds, and are dissociated to polyanion withnegative net charges at pH above 2.0, so the anion exchanger cantheoretically be used for nucleic acid extraction, concentration andanalysis. Conventional anion exchangers have high adsorption capacityfor nucleic acids, but how to elute the adsorbed nucleic acids istechnically challenging. A monovalent neutral inorganic salt at highconcentrations can be used to improve the elution efficacy of theadsorbed nucleic acid anions, while the excessive inorganic salt mayinhibit the subsequent PCR amplification. Currently, the rapidextraction of nucleic acids in the samples for analysis uses silanol asan adsorption group by forming hydrogen bonds under mild conditions toensure the elution efficacy. In addition, in order to improve thereproducibility of nucleic acid extraction to ensure the reproducibilityof nucleic acid analysis, micro-magnetic beads are commonly used forrapid adsorption, separation and elution of nucleic acids. However, thesilanol has low nucleic acid-binding capacity and low selectivity tonucleic acids, resulting in the use of a large amount of silanolmagnetic beads at high cost. Many impurities may also be adsorbed andeluted in the nucleic acid elution due to high non-specific adsorptionof the surface of silanol magnetic beads, thereby inhibiting the PCR andaffecting the sensitivity of nucleic acid detection. Ion exchange isalso a conventional method for protein purification, but in thisapplication, the target protein often has a low elution yield even if amonovalent neutral inorganic salt is used at a high concentration forcompetitive elution. Moreover, the inorganic salt requires to be removedby dialysis or ultrafiltration before subsequent operations/analyses,reducing the operation efficiency and increasing the cost. Therefore,there is a need to develop novel ion exchangers for rapidextraction/purification and preparation of nucleic acids and proteins toimprove the elution efficacy of the adsorbed substance with a lowionic-strength buffer.

After the target substance is adsorbed on the surface of the ionexchange medium by electrostatic attraction, the ion exchange medium canbe treated by an eluent with significantly different pH to have asurface net charge of zero, thereby enhancing the elution efficacy ofthe target substance. However, a large amount of hydrogen bonds betweenthe target substance and the ion exchange medium still retard theelution, and the non-specific adsorption of common ion exchange mediumsto interfering small molecules on common ion exchange media mayinterfere with the subsequent analyses. The generation of electrostaticrepulsion between the adsorbed target substance and the ion exchangemedium can certainly promote the elution of the adsorbed targetsubstance and the regeneration of the separation medium. An eluent withpH significantly different from that of an adsorption buffer is expectedto generate electrostatic repulsion between the target substance and thesurface of the separation medium. However, there is only one type ofdissociable groups on the surface of any one classic ion exchanger, sothat the opposite charge between the net charge on the surface of theseparation medium and the net charge of the target substance cannot beeasily changed to the same by adjusting the elution pH, thereby failingto generate electrostatic repulsion and promote elution.

Therefore, in order to overcome the defects of classical ion exchangeseparation medium in the separation of biomolecules, there is a need todevelop a novel ion exchange medium that provides high adsorptioncapacity, high elution efficacy of the adsorbed substance under mildconditions, separation capacity which can be accurately and easilycalibrated, low non-specific adsorption of non-target substances andhigh regeneration efficacy.

SUMMARY

In view of the above, the application provides a novel ion exchangemedium with high-efficiency adsorption, large adsorption capacity,high-efficacy elution of adsorbed ions under mild conditions, easyaccurate calibration of separation capacity, low non-specific adsorptionof non-target substances and high regeneration ability under mildconditions.

The application provides an amphoteric dissociation ion exchangeseparation medium, wherein a surface of the amphoteric dissociation ionexchange separation medium is an amphoteric dissociationcovalently-modified layer; the amphoteric dissociationcovalently-modified layer has an isoelectric point, denoted as pIm, andthe isoelectric point is an environmental pH value at which the netcharge on the surface of the amphoteric dissociation ion exchangeseparation medium is zero; wherein, when the environmental pH value islower than the pIm, the net charge on the surface of the amphotericdissociation covalently-modified layer is positive and the amphotericdissociation ion exchange separation medium has the properties of ananion exchanger; when the environmental pH value is higher than the pIm,the net charge on the surface of the amphoteric dissociationcovalently-modified layer is negative and the amphoteric dissociationion exchange separation medium has the properties of a cation exchanger.

In an embodiment, when the environmental pH value is lower than the pIm,the number of positive net charge on the surface of the amphotericdissociation covalently-modified layer gradually increases as thedifference between the pIm and the environmental pH value increases;when the environmental pH value is higher than the pIm, the number ofnegative net charge on the surface of the amphoteric dissociationcovalently-modified layer gradually increases as the difference betweenthe pIm and the environmental pH value increases. Clearly, theenvironmental pH value is located in a pH range which is tolerated byboth a target substance to be separated and the amphoteric dissociationion exchange separation medium.

In an embodiment, the amphoteric dissociation covalently-modified layeron the surface of the amphoteric dissociation ion exchange separationmedium simultaneously comprises a group which dissociates to generatepositive charge when pH is lower than the pIm, and a group whichdissociates to generate negative charge when pH is higher than the pIm;and the group which dissociates to generate negative charge is analiphatic carboxyl group, and the group which dissociates to generatepositive charge is one or more groups selected from the group consistingof an aliphatic primary amine group, an aliphatic secondary amine group,an aliphatic tertiary amine group and an imidazolyl group.

In an embodiment, the amphoteric dissociation covalently-modified layeris derived from covalent modification of the surface of a separationmedium substrate by an amphoteric dissociation group precursor;

the surface of the separation medium substrate does not contain a linearlong chain having a length more than 9 atoms, but contains a reactivegroup for covalently linking with the amphoteric dissociation groupprecursor to form the amphoteric dissociation covalently modified layer;the reactive group is any one group selected from the group consistingof an aliphatic primary amine group, an aliphatic secondary amine group,an aliphatic carboxyl group and a thiol reactive group; wherein thethiol reactive group contains a small-size group that is substituted bya thiol and leaves from the surface of the separation medium substrate;

the amphoteric dissociation group precursor is a hydrophilic materialhaving a molecular weight less than 500 Daltons and no hydrocarbonchains or hydrocarbon rings with more than 5 successive carbon atoms;wherein the amphoteric dissociation group precursor contains analkylthiol group or an aliphatic primary amine group as acovalently-linking group and an aliphatic carboxyl group as a groupwhich dissociates to generate negative charge, and/or one or more groupsselected from the group consisting of an aliphatic primary amine group,an aliphatic secondary amine group, an aliphatic tertiary amine groupand an imidazolyl group as a group which dissociates to generatepositive charge; the amphoteric dissociation group precursor comprises atype A precursor, a type B precursor and a type C precursor, wherein thetype A precursor is an amphoteric dissociation group precursorcomprising only the group which dissociates to generate negative chargebesides the covalently-linking group, the type B precursor is anamphoteric dissociation group precursor comprising only the group whichdissociates to generate positive charge besides the covalently-linkinggroup, and the type C precursor is an amphoteric dissociation groupprecursor comprising the group which dissociates to generate negativecharge and the group which dissociates to generate positive chargebesides the covalently-linking group; and the separation mediumsubstrate is covalently modified with the amphoteric dissociation groupprecursor by one of the following methods:

a) using the type C precursor alone or in combination; wherein when usedin combination, various type C precursors are mixed at a given ratio foruse, and the precursors for mixing contain the same covalently-linkinggroup;

b) mixing one or more type A precursors with one or more type Bprecursors at a given ratio for use; wherein these precursors for mixingcontain the same covalently-linking group;

c) mixing only one or more type A precursors with one or more type Cprecursors in a given ratio; wherein these precursors for mixing containthe same covalently-linking group; and

d) mixing only one or more type B precursors with one or more type Cprecursors in a given ratio; wherein these precursors for mixing containthe same covalently-linking group.

In an embodiment, when the amphoteric dissociation group precursor isused to covalently modify the separation medium substrate, a molar ratioof the total amount of aliphatic primary, secondary, tertiary aminegroups and imidazolyl groups to the total amount of aliphatic carboxylgroups in the amphoteric dissociation group precursor is limited to 1:6to 6:1.

In an embodiment, when an aliphatic secondary amine and/or an aliphatictertiary amine are/is used in an amphoteric dissociation group precursoras the group which dissociates to generate positive charges, the totalmolar amount of the aliphatic secondary amine and the aliphatic tertiaryamine does not exceed 30% of the total molar amount of the aliphaticprimary amine and the imidazolyl group in precursors used.

In an embodiment, the number of positive charges on the surface of theamphoteric dissociation ion exchange separation medium is not less than90% of the maximum value at pH 3, and the number of negative charge onthe surface of the amphoteric dissociation ion exchange separationmedium is not less than 90% of the maximum value at pH 11.

The application further discloses a use method of the amphotericdissociation ion exchange separation medium, comprising:

applying the amphoteric dissociation ion exchange separation medium toseparate a target substance; wherein a common logarithm of adissociation constant obtained from the release of hydrogen ion from thetarget substance is pK or an isoelectric point of the target substanceis pI; wherein the method further comprises:

a. selecting an amphoteric dissociation ion exchange separation medium;wherein the difference between the pIm of the amphoteric dissociationion exchange separation medium and the pK or pI of the target substanceis not less than 1.0;

b. adsorbing the target substance; wherein an environment pH is selectedto allow the type of net charges on the surface of the ion exchangeseparation medium to be opposite to the type of net charges of thetarget substance, thereby resulting in adsorption of the targetsubstance by electrostatic attraction, wherein the environmental pH islocated between the pK or pI of the target substance and the pIm of theamphoteric dissociation ion exchange separation medium and thedifference between the environmental pH and the pK or pI of the targetsubstance and the difference between the environmental pH and the pIm ofthe amphoteric dissociation ion exchange separation medium are bothgreater than 0.3; and

c. eluting the target substance; wherein when pH of an eluent is lowerthan the pIm of the amphoteric dissociation ion exchange separationmedium, the pH of the eluent is at least 0.3 lower than the lower one ofthe pK or pI of the target substance and the pIm of the amphotericdissociation ion exchange separation medium; when the pH of the eluentis higher than the pIm of the amphoteric dissociation ion exchangeseparation medium, the pH of the eluent is at least 0.3 higher than thehigher one of the pK or pI of the target substance and the pIm of theamphoteric dissociation ion exchange separation medium; in the use ofthe above eluent, the type of net charges of the surface of theamphoteric dissociation ion exchange separation medium is the same asthat of the target substance, thereby resulting in elution of the targetsubstance by electrostatic repulsion; and a water-soluble monovalentneutral inorganic salt is added to the eluent to promote the elution ofthe target substance.

The application also discloses a method for calibrating separationcapacity of the amphoteric dissociation ion exchange separation medium,comprising:

a) selecting an colored organic compound as a color-developing probe forthe calibration of the separation capacity of the amphotericdissociation ion exchange separation medium; wherein the colored organiccompound has a dissociation constant of pK or an isoelectric point ofpI, a molecular weight less than 600 Daltons, a visible light absorptioncoefficient greater than 14 mM⁻¹. cm⁻¹, a solubility not less than 5.0μmol/L at pH 3.0-11.0, and a positive or negative net charge afterdissociation at pH 3.0-11.0;

b) calibrating the separation capacity of the amphoteric dissociationion exchange separation medium; wherein step b comprises:

b1) when the amphoteric dissociation ion exchange separation medium hasan isoelectric point pIm between 4.0 and 6.0, using a cationic probewith a dissociation constant of pK or an isoelectric point of pI atleast 2.0 greater than the pIm of the amphoteric dissociation ionexchange separation medium, or using an anionic probe with adissociation constant of pK or an isoelectric point of pI at least 2.0lower than the pIm of the amphoteric dissociation ion exchangeseparation medium; and

b2) when the amphoteric dissociation ion exchange separation medium hasthe pIm between 6.0 and 10.0, using the anionic probe with adissociation constant of pK or an isoelectric point pI at least 2.0lower than the pIm of the amphoteric dissociation ion exchangeseparation medium;

c) when calibrating the separation capacity of the amphotericdissociation ion exchange separation medium, selecting a buffer solutioncorresponding to an environmental pH to allow the amphotericdissociation ion exchange separation medium to adsorb a color-developingprobe by electrostatic attraction, wherein the environmental pH isbetween the dissociation constant pK or the isoelectric point pI of thecolor-developing probe and the pIm of the amphoteric dissociation ionexchange separation medium, and differs from the pIm of the amphotericdissociation ion exchange separation medium by not less than 1.3 anddiffers from the dissociation constant pK or the isoelectric point pI ofthe color-developing probe by not less than 0.5; selecting a buffersolution corresponding to an environmental pH to allow the type of netcharges on the surface of the amphoteric dissociation ion exchangeseparation medium to be the same as that of the color-developing probein the elution, thereby eluting a color-developing probe byelectrostatic repulsion, wherein the environmental pH is at least 1.3higher than the higher one or at least 1.3 lower than the lower one ofthe dissociation constant pK or the isoelectric point pI of thecolor-developing probe and the pIm of the amphoteric dissociation ionexchange separation medium; measuring the adsorption of acolor-developing probe in an eluate followed by conversion into aseparation capacity for the color-developing probe; and

determining pH effect on the separation capacity of the amphotericdissociation ion exchange separation medium to a color-developing probe;wherein the minimum pH at which the separation capacity for thecolor-developing probe with negative net charge reaches zero, or themaximum pH at which the separation capacity for the color-developingprobe with positive net charge reaches zero, is an approximation of thepIm of the amphoteric dissociation ion exchange separation medium.

The application has the following beneficial effects.

The invention provides an amphoteric dissociation ion exchangeseparation medium, where covalent modification is adopted to form astructure of amphoteric dissociation covalently-modified layer on thesurface of the medium and a specific isoelectric point pIm is formedaccording to the difference in the modified structure or the proportionof the amphoteric dissociation group precursors. In addition, theseparation medium has the properties of an anion exchanger and a cationexchanger, respectively, at different environmental pHs on both sides ofthe pIm. The ion exchange property of the amphoteric dissociation ionexchange separation medium can be reversed by changing a pH at one sideof the pIm into a pH at the other side of the pIm, thereby providingsignificantly different adsorption and separation effect on the chargedsubstance as the pH crosses the pIm. pH of a buffer solution can beadjusted to allow the surface of the amphoteric dissociation ionexchange separation medium of the invention and the target substance tohave the same net charge, so that the target substance can be releasedby electrostatic repulsion, thereby enhancing the elution efficacy,especially increasing the elution rate and improving the regenerationproperty of the separation medium, and avoiding the subsequent problemcaused by using high-concentration inorganic ions. The competitivebinding between ions is no longer necessary in the elution, but canstill be used to promote the elution. In summary, the amphotericdissociation ion exchange separation medium of the invention has bothelectrostatic interaction-based adsorption and electrostaticinteraction-based elution performances, and also provides highseparation capacity, high-efficacy elution of adsorbed ions under mildconditions, easy accurate calibration of separation capacity, lownon-specific adsorption to non-target substances and high regenerativeefficiency under mild conditions. Furthermore, the invention employsspecial amphoteric dissociation group precursors to covalently modify aseparation medium substrate to form the desired amphoteric dissociationcovalently-modified layer, allowing the separation medium surface for anamphoteric dissociation property. This formation method has thefollowing beneficial effects. First, this strategy makes it easier toadjust the molar ratio of a group dissociating to form an anion to agroup dissociating to form a cation to obtain different pIm favorablefor the adsorption of different biomolecules. It is apparent that thisstrategy is simpler and more practical, and involves higher efficiencywhen compared to the direct preparation of a separation medium with anamphoteric dissociation group on the surface by adjusting thepolymerization conditions. Secondly, a natural cellulose-polymerseparation medium is suitable as a substrate to prepare an amphotericdissociation ion exchange separation medium by such a method. It shouldbe noted that the existing technology fails to prepare the cellulose bypolymerization, let alone directly prepare a cellulose with anamphoteric dissociation group on the surface by polymerization.Furthermore, this method facilitates the selection of a separationmedium satisfying specific requirements as a substrate for covalentmodification to obtain the amphoteric dissociation surface, which isadvantageous for avoiding reduction in coverage of amphiphilicdissociation groups on the surface caused by steric hindrance betweencharged groups during polymerization, thereby ensuring high coverage ofhydrophilic groups on the surface and reducing non-specific adsorptionto hydrophobic small molecules. In other words, this formation methodplays two important roles respectively in forming the desired amphotericdissociation group on the surface and in reducing non-specificadsorption of the surface to hydrophobic substances, allowing theamphoteric dissociation ion exchange separation medium for a betterperformance.

The application further discloses a use method of the amphotericdissociation ion exchange separation medium and a method for calibratingthe separation capacity of the amphoteric dissociation ion exchangeseparation medium. The use method is simple and only requires acolor-developing probe with a specific pK or pI. The process ofcalibrating the separation capacity is simple and rapid, and themeasurement of the absorbance of the probe is easy to be standardizedand has good reproducibility. Therefore, this calibration method issuitable for the standard setting and tracing of product quality andcharacterization of the amphoteric dissociation property of theamphoteric dissociation ion exchange separation medium, therebyproviding rapid, simple and high-accuracy calibration of the separationcapacity of the ion exchange separation medium and characterization ofits properties.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below with reference tothe drawings and embodiments.

FIG. 1 shows the change in surface net charge of FBD-MSP-ZEW with pH.

FIG. 2 schematically shows the process of converting FBD-MSP-FCOOH intoan active ester.

FIG. 3 shows the formation of an amphoteric dissociationcovalently-modified layer from an FBD-MSP-FCOOH active ester by formingan amide bond.

FIG. 4 shows the formation of an amphoteric dissociationcovalently-modified layer by converting FBD-MSP-FCOOH active ester intoa thiol reactive group.

FIG. 5 shows the assay of an extinction coefficient of acid red 13 at pH8.3.

FIG. 6 shows response of the amount of acid red 13 adsorbed andseparated to the amount of a reaction system FBD-MSP-ZEWA;

FIG. 7 shows response of the amount of acid red 13 adsorbed andseparated to the concentration of a reaction system acid red 13.

FIG. 8 shows the effect of pH of the adsorption on the capacity of threeamphoteric dissociation magnetic beads to separate acid red 13 and theestimation of pIm of the three magnetic beads.

FIG. 9 shows the effect of the amount of plasmid in the adsorptionsystem on the amount of plasmid obtained by magnetic bead separation.

FIG. 10 shows response of the amount of plasmid separated to the amountof magnetic beads in the case of excess plasmid.

FIG. 11 shows semi-quantitative comparison of the efficacy of PCR afterthe plasmid is separated with magnetic beads and recycled in the casethat the plasmid is not excessive.

FIG. 12 shows SDS-PAGE analysis of the purification effect ofFBD-MSP-ZEWB on Pichia guilliermondii uricase RMGU. M: protein molecularweight Marker; 1: natural MGU, treated by DEAE-cellulose adsorptionthree times for removal of other proteins and purification and 5 μg forloading; 2: cell lysate, with a total protein of 5 μg for loading; 3: alyophilized product of a second eluate obtained by elution with 0.20 mLof crude enzyme, 5 μg for loading; 4: a lyophilized product of a secondeluate obtained by elution with 1.0 mL of crude enzyme, 5 μg forloading; 5: a lyophilized product of a second eluate obtained by elutionwith 4.0 mL of crude enzyme, 5 μg for loading; 6: a crude enzyme, with atotal protein of 15 μg for loading; and 7: a crude enzyme, with a totalprotein of 45 μg for loading.

FIG. 13 shows the detection and comparison of fluorescence real-timequantitative PCR procedure for detecting interference from blankmagnetic beads (see Example 7 for plasmid samples).

FIG. 14 shows the detection and comparison of fluorescence real-timequantitative PCR procedure for detecting the extraction of plasmids bymagnetic beads (see Example 7 for plasmid samples).

FIG. 15 shows the MGU expression plasmid response curve detected byfluorescence real-time quantitative PCR.

DETAILED DESCRIPTION OF EMBODIMENTS

Provided in the embodiments is an amphoteric dissociation ion exchangeseparation medium, where the surface of the amphoteric dissociation ionexchange separation medium is an amphoteric dissociationcovalently-modified layer; the amphoteric dissociationcovalently-modified layer has an isoelectric point (pIm), and the pIm isan environmental pH value at which the net charge on the surface of theamphoteric dissociation ion exchange separation medium is zero; when theenvironmental pH value is lower than the pIm, the net charge on thesurface of the amphoteric dissociation covalently-modified layer ispositive and the amphoteric dissociation ion exchange separation mediumhas the properties of an anion exchanger; when the environmental pHvalue is higher than the pIm, the net charge on the surface of theamphoteric dissociation covalently-modified layer is negative and theamphoteric dissociation ion exchange separation medium has theproperties of a cation exchanger. The amphoteric dissociationcovalently-modified layer contains an amphoteric dissociation substancewhich contains both a group dissociating to generate negative charge anda group dissociating to generate positive charge. The electrostaticattraction between the separation medium bearing an amphotericdissociation covalently-modified layer on the surface and the targetsubstance can be reversed to electrostatic repulsion using adsorptionand elution buffer solutions with a significant difference in pH,enabling efficient elution based on electrostatic repulsion. In a pHrange that the separation medium and the target substance can tolerate,this novel elution based on electrostatic repulsion can be performed byadjusting the pH to change the net charge type of the surface of theseparation medium and keep the net charge type of the target substanceunchanged, or to change the net charge type of the target substance andkeep the net charge type of the separation medium unchanged. The pHrange that the bioactive substance can tolerate is limited. Unless thetarget substance is an amphoteric dissociation substance, its chargetype cannot be reversed, thereby failing to reverse the electrostaticinteraction between the target substance and the separation mediumsurface from attraction to repulsion. In order to make it applicable forboth the amphoteric dissociation target substance and the non-amphotericdissociation target substance, there is a need to develop a novel ionexchange separation medium of which the net charge type can be changedby adjusting pH in a limited range, that is, an ion exchange separationmedium with an amphoteric dissociation modification layer on its surface(as shown in FIG. 1).

The charge type of an amphoteric dissociation substance is oftencharacterized by a pH at which its net charge is zero, that is, theisoelectric point pI, while the charge type of a non-amphotericdissociation substance is often characterized by its dissociationconstant pK. For convenience, the isoelectric point of the amphotericdissociation target substance is also called pIs and a pH at which thesurface net charge of the ion exchange separation medium is zero, i.e.,the isoelectric point, is called pIm. Stronger hydrophilicity of thesurface of the ion exchange separation medium may result in lowernon-specific adsorption to most substances, especially the unchargedhydrophobic substances, which is beneficial to improve the regenerationefficacy and separation selectivity of such separation medium, therebyimproving the sensitivity of subsequent analysis. However, to achievethe reversal from efficient adsorption of the target substance to theseparation medium surface based on electrostatic attraction to efficientelution of the target substance from the separation medium surface basedon electrostatic repulsion by adjusting pH, the pIm of the amphotericdissociation ion exchange separation medium is also required to meet thefollowing conditions: in a pH range which the target substance and theseparation medium can tolerate, the net charge of the separation mediumsurface is positive at a lower pH and can be reversed to negative at ahigher pH, which indicates that the pIm of the separation medium isrequired to be in the middle of the pH range which both the targetsubstance and the separation medium can tolerate; in addition, for anamphoteric dissociation target substance, it is also required that thereis sufficiently large difference between the pIm of the suitable ionexchange separation medium and the pIs of the target substance. Aftermeeting such conditions, pH of the adsorption buffer is selected toallow the net charge of the target substance to be opposite to that ofthe separation medium surface to provide electrostatic adsorption,thereby minimizing the non-specific adsorption of interfering substancesby washing the separation medium after the adsorption of the targetsubstance by the adsorption buffer. Then, a buffer with a suitable pH isused to allow the net charge of the target substance to be the same asthat of the separation medium surface to provide electrostaticrepulsion, thereby efficiently eluting the target substance. Awater-soluble monovalent neutral inorganic salt may be added to promotethe elution. This ion exchange separation medium exhibits significantadvantages in extracting and concentrating a charged target substance,so that it may have a longer life and higher separation efficacy whenused as an ion exchange chromatography column packing material, orprovide higher regeneration efficacy, simple and rapid separation andhigher yield of the target substance when used in the preparation of thetarget substance.

The adsorption and elution of the target substance on the surface of theion exchange separation medium are achieved by adjusting pH, where theprinciple of the elution in the use of this separation medium issignificantly different from that in the use of the classical ionexchanger. In the use of such ion exchange separation medium, thesurface of the separation medium and the target substance can have thesame type of net charges by adjusting the elution pH, so thatelectrostatic repulsion is generated to promote the release of thetarget substance, thereby improving the elution efficacy, especiallyincreasing the elution rate and elution percentage of the adsorbedtarget substance, and avoiding the subsequent problems caused byhigh-concentration inorganic ions. In the elution, the competitivebinding between ions is no longer necessary, but can still be introducedto promote the elution. The adsorption and separation of the targetedsubstance with the amphoteric dissociation ion exchange separationmedium at a pH which is at one side of the pIm are significantlydifferent from those at a pH which is at the other side of the pIm.Specifically, the separation medium adsorbs anions at a pH lower thanthe pIm, that is, playing a role as an anion exchanger; the separationmedium adsorbs cations at a pH higher than the pIm, that is, playing arole as a cation exchanger. Obviously, the adsorption of the chargedsubstance on the classical ion exchanger cannot be reversed by theadjustment of pH.

When the environmental pH value is lower than the pIm of the amphotericdissociation ion exchange separation medium, the number of positivecharges on the surface of the amphoteric dissociationcovalently-modified layer gradually increases as the difference betweenthe pIm and the environmental pH value increases; when the environmentalpH value is higher than the pIm, the number of negative charges on thesurface of the amphoteric dissociation covalently-modified layergradually increases as the difference between the pIm and theenvironmental pH value increases. The environmental pH value is locatedin a pH range which is tolerated by both a target substance and theamphoteric dissociation ion exchange separation medium.

In this embodiment, the amphoteric dissociation covalently-modifiedlayer on the surface of the amphoteric dissociation ion exchangeseparation medium comprises simultaneously a group which dissociates togenerate positive charge when pH is lower than the pIm, and a groupwhich dissociates to generate negative charge when pH is higher than thepIm. The group which dissociates to generate negative charge is analiphatic carboxyl group, and the group which dissociates to generatepositive charge is one or more groups selected from the group consistingof an aliphatic primary amine group, an aliphatic secondary amine group,an aliphatic tertiary amine group and an imidazolyl group.

In this embodiment, the amphoteric dissociation covalently-modifiedlayer is derived from covalent modification of the surface of aseparation medium substrate by an amphoteric dissociation groupprecursor.

The separation medium substrate is a particle of any size or a film ofany thickness that does not contain a linear long chain having a lengthmore than 9 atoms, but contains any one of the following reactive groupsfor covalently linking with the amphoteric dissociation group precursorsto form the amphoteric dissociation covalently modified layer: analiphatic primary amine group, an aliphatic secondary amine group, analiphatic carboxyl group and a thiol reactive group; where the thiolreactive group contains a small-size group that is substituted by athiol and leaves from the surface of the separation medium substrate.

The amphoteric dissociation group precursor is a hydrophilic materialhaving a molecular weight less than 500 Daltons and no hydrocarbonchains or hydrocarbon rings with more than 5 successive atoms. Theamphoteric dissociation group precursor contains an alkylthiol group oran aliphatic primary amine group for covalent linking and an aliphaticcarboxyl group as a group which dissociates to generate negative charge,and/or one or more groups selected from the group consisting of analiphatic primary amine group, an aliphatic secondary amine group, analiphatic tertiary amine group and an imidazolyl group, as a group whichdissociates to generate positive charge. The amphoteric dissociationgroup precursor comprises a type A precursor, a type B precursor and atype C precursor. The type A precursor is an amphoteric dissociationgroup precursor comprising only the group which dissociates to generatenegative charge besides the covalently-linking group. The type Bprecursor is an amphoteric dissociation group precursor comprising onlythe group which dissociates to generate positive charge besides thecovalently-linking group. The type C precursor is an amphotericdissociation group precursor comprising the group which dissociates togenerate negative charge and the group which dissociates to generatepositive charge besides the covalently-linking group. The type Cprecursor comprises lysine, ornithine, histidine,N,N-dicarboxymethylethylenediamine, cysteine, 3-thiolhistidine, 3-thiollysine and 3-thiol glutamic acid; the type A precursor comprisesglutamic acid, 3-thiol-1,5-glutaric acid, thioglycolic acid andtris-(carboxymethyl)-aminomethane; and the type B precursor comprisesmercaptoethylamine, 3-thiol-2-hydroxypropylamine, 2-mercaptoimidazole,diethylenetriamine, N,N-dimethylaminoethylenediamine andtetra-(aminomethyl)-methane. These substances may be used alone or incombination of two or more of them to adjust the ratio of the groupwhich dissociates to generate positive charges to the group whichdissociates to generate negative charges, thereby adjusting pIms of theamphoteric dissociation ion exchange separation mediums.

The separation medium substrate is covalently modified with theamphoteric dissociation group precursor(s) by one of the followingmethods:

a) using the type C precursor alone or in combination; wherein when usedin combination, various type C precursors are mixed at a given ratio foruse, and the precursors for mixing contain the same type of groups forcovalent linking;

b) mixing one or more type A precursors with one or more type Bprecursors in a given ratio for use; wherein these precursors for mixingcontain the same type of groups for covalent linking;

c) mixing only one or more type A precursors with one or more type Cprecursors in a given ratio; wherein these precursors for mixing containthe same type of groups for covalent linking; and

d) mixing only one or more type B precursors with one or more type Cprecursors in a given ratio; wherein these precursors for mixing containthe same type of groups for covalent linking.

When the amphoteric dissociation group precursor is used to covalentlymodify the separation medium substrate, the molar ratio of the totalamount of aliphatic primary, secondary, tertiary amine groups andimidazolyl groups to the total amount of aliphatic carboxyl groups inthe amphoteric dissociation group precursor(s) is limited to 1:6 to 6:1.When an aliphatic secondary amine and/or an aliphatic tertiary amineare/is used in an amphoteric dissociation group precursor as the groupwhich dissociates to generate positive charge, the total molar amount ofthe aliphatic secondary amine and the aliphatic tertiary amine does notexceed 30% of the total molar amount of the aliphatic primary amine andthe imidazolyl group.

There are two kinds of covalent-linking reactions: amide formationbetween the aliphatic amine group and the aliphatic carboxyl group, andsubstitution of a leaving group by an alkyl thiol.

There is no amphoteric dissociation covalently-modified layer and nolong linear groups with more than 9 atoms on the surface of theseparation substrate. When the separation medium without the amphotericdissociation covalently-modified layer is used as the substrate, thereactive group on its surface is one of an aliphatic carboxyl group, analiphatic primary amine group, an aliphatic secondary amine group and athiol reactive group. The thiol reactive group contains a small-sizeleaving group which can be easily and nucleophilically substituted by athiol group and leaves the surface of the separation medium. Such asmall-size leaving group includes a halogen ion, p-toluenesulfonategroup and a trifluoroacetate group. When such reactive groups are absenton the surface of the separation medium without the amphotericdissociation covalently-modified layer, the separation medium can beprovided with these reactive groups by appropriate derivatization to bea separation medium substrate. Then the separation medium substrate isconverted into the amphoteric dissociation ion exchange separationmedium by modification with an amphoteric dissociation group precursor.Cellulose or hydrophilic macroporous resin with hydroxyl groups on thesurface as a separation medium can be modified/derived with succinicanhydride or chloroacetic anhydride to obtain the desired reactivegroup, so that the modified cellulose or macroporous resin is suitableas a substrate for further formation of the amphoteric dissociationcovalently-modified layer.

When the separation medium without the amphoteric dissociationcovalently-modified layer is used as the substrate and an aliphaticprimary amine group and/or an aliphatic secondary amine group are/isprovided on the surface as reactive groups for covalent linking, theamine group reacts with a halogenated acetic anhydride or a haloaceticacid active ester and then reacts with a thiol-containing amphotericdissociation group precursor, or substitutes the halogen in bromaceticacid or iodoacetic acid to form glycine, or reacts with succinicanhydride or a mixture of succinic anhydride and acetic anhydride in thecase that the total molar amount of the acid anhydride is not excessive,to form a covalently-modified layer composed of an amphotericdissociation group.

In this embodiment, the number of positive charges on the surface of theamphoteric dissociation ion exchange separation medium is not less than90% of the maximum value at pH 3, and the number of negative charges onthe surface of the amphoteric dissociation ion exchange separationmedium is not less than 90% of the maximum value at pH 11.

The adsorption and elution of a target substance are performed with anion exchange separation medium by adjusting pH, where the surface ofthis ion exchange medium is required to be an amphoteric dissociationmodification layer, which is significantly different from the surfacestructure of the classical ion exchange medium. The separationperformance of the separation medium can only be maximized in the casethat the number of positive charges and the number of negative chargeson the surface of the ion exchange separation medium are close to theirmaximum values respectively at two boundaries of the pH range which boththe target substance and the separation medium can tolerate. When thenumber of the net charges on the surface of the ion exchange separationmedium is not less than 90% of the maximum value, the separationperformance can be approximately maximized. The hydrophilic group issuitable for the preparation of the amphoteric dissociation ion exchangeseparation medium, while the aromatic substance is unsuitable as anamphoteric dissociation group precursor. Common groups which ionize togenerate negative charges include a phosphomonoester, a phosphodiester,an organic sulfonic acid and an aliphatic carboxylic acid, where thephosphomonoester, the phosphodiester and the organic sulfonic acid allhave pKs less than 1.7; and for the carboxylic acid with an isolatedcarboxyl group, such as acetic acid, the pK of its carboxyl group isnear 4.8. In addition, imidazole is hydrophilic and has a pK near 6.0;an isolated primary amine with hydrophilic substituents, such as tris(hydroxymethyl) aminomethane, has a pK near 8.0; methylamine anddimethylamine have a pK near 10.7 and the pK of trimethylamine is near10.0; and ethylamine, diethylamine and triethylamine all have pKs near11.0. In another aspect, the spatial neighboring dissociable groups inthe amphoteric dissociation modification layer have a significant effecton the respective dissociation constants. For example, the pK of aceticacid is near 4.8 and the pK of ethylamine is near 11.0; tris(hydroxymethyl) aminomethane comprises a neural group adjacent to theamine group and its pK is near 8.0, which is much lower than the pK ofethylamine (11.0); and glycine and alanine both contain equimolaramounts of adjacent aliphatic carboxy group and primary amine group,with the pK near 6.0 and much lower than the mean value (7.8) of the pKof ethylamine and the pK of acetic acid. In addition, there is a greatdifference in pKs of the carboxyl groups in EDTA or EGTA, and a greatdifference is also observed between the pKs of the two amine groups inethylenediamine. If the spatial distribution of the group whichdissociates to generate positive charge and the group which dissociatesto generate negative charge is random in the amphoteric dissociationcovalently-modified layer, the pK of each of the two types ofdissociable groups is significantly lower than that of the isolatedcorresponding group. If the group which dissociates to generate positivecharge or the group which dissociates to generate negative charge isclustered and agglomerated, an apparent pK resulting from its fullionization will be higher. The group which dissociates to generatepositive charges has a dissociation degree lower than 10% at a pH 1.0unit higher than pK, and the group which dissociates to generatenegative charges both has a dissociation degree lower than 10% at a pH1.0 unit lower than pK. A common and simplest method for preparing theamphoteric dissociation ion exchange separation medium of theapplication is to use a group dissociating to generate negative chargewhich has an ionization degree as far as possible lower than 10% at pH4.0, and a group dissociating to generate positive charge which has anionization degree as far as possible lower than 10% at pH 10.0, thus,the separation performance of the separation medium can be maximizedbetween pH 3.0 and pH 11.0. In view of this, the pK of the groupdissociating to generate positive charge is required to be as far aspossible greater than 4.0, and only an aliphatic carboxylic acid with aplurality of spatially adjacent carboxyl groups can provide a higherapparent pK due to the full ionization of its carboxyl groups; the pK ofthe group dissociating to generate negative charges is required to be asfar as possible lower than 9.0, and only an imidazole group and analiphatic primary amine group having spatially adjacent substituentswhich dissociate to generate negative charges or are neutral andhydrophilic can meet the requirements, and the aliphatic primary aminegroup is required to be separated as far as possible by the aliphaticcarboxyl group to reduce its pK. The secondary and tertiary aminessubstituted with an ethyl or a larger alkyl have higher pK and stronghydrophobicity, so that they are not suitable for the preparation of theseparation medium. Even if a methyl-substituted aliphatic secondary ortertiary amine is used to adjust the pIm, the molar ratio of which tothe total of aliphatic primary amine and imidazole should be as low aspossible. Theoretically, the separation performance of the separationmedium having a covalently-modified layer composed of the aboveamphoteric dissociation groups on the surface can be maximized at a pHbetween 3.0 and 11.0.

Obviously, the molar ratio of the group dissociating to generatenegative charge to the group dissociating to generate positive chargenot only determines the proportion of the positively charged aliphaticprimary amine/imidazole group relative to the nearly fully-ionizedaliphatic carboxyl group at pH 11.0, but also determines the proportionof the negatively charged aliphatic carboxyl group relative to thenearly fully-ionized aliphatic primary amine/imidazole group at pH 3.0,that is, the maximum separation performance depends on whether the netcharge on the surface of the separation medium is nearly maximized atthe boundaries of this pH range. It is apparent that a ratio between thetwo types of dissociable groups on the surface of the amphotericdissociation separation medium of the invention is required to beoptimized. At a pH 3 units lower than the pK of an acidic dissociablegroup or 3 units greater than the pK of a basic dissociable group, thedissociation/ionization degree of the acidic or basic dissociable groupis lower than 0.1%. It is reasonable to assume that the differencebetween the pK of the adjacent aliphatic carboxyl group and aliphaticprimary amine group is still much greater than 2.5. When at a pH 1 unithigher than the pK of the above group dissociating to generate positivecharges, the dissociation degree of the aliphatic carboxyl group togenerate negative charges may exceed 99% and the ionization degree ofthe group dissociating to generate positive charge is lower than 10%.When at a pH 1 unit lower than the pK of the group dissociating togenerate negative charges, the dissociation degree of the aliphaticcarboxyl group to generate negative charges is lower than 10% and thedissociation degree of the above group dissociating to generate positivecharges may exceed 99%. It is assumed that an ionization degree of thealiphatic carboxyl group on the surface of the amphoteric dissociationseparation medium of the present invention to dissociate to generate anegatively charged group is lower than 2% at pH 3.0 due to the apparentpK near 4.8. At pH 11.0, the ionization degree of the aliphatic carboxylgroup to dissociate to generate a positively charged group is lower than1% since the pK of the primary amine with a hydrophilic aliphaticsubstituent is less than 9.0 and contents of the aliphatic secondary andtertiary amines are negligible, and in such case, as long as the molarratio of the aliphatic carboxyl group to the total of the aliphaticprimary amine group and the imidazolyl group is 1:6 to 3:1, theseparation performance may be nearly maximized between pH 3.0 and pH11.0. Given that a higher density of the surface aliphatic carboxylgroup may allow more carboxyl groups to be adjacent so that the pK ofthe carboxyl group may be increased and the adjacency between thecarboxyl group and the amine group may lower the pK of the amine group,the present invention limits the ratio of the aliphatic primary aminegroup (the total of the secondary amine and the tertiary amine accountsfor not higher than 30% of the group dissociating to generate positivecharge) to the aliphatic carboxyl group to 1:6 to 6:1 to enable themaximization of the separation performance at pH 3.0 to 11.0. A mixtureof the two types of precursors containing the group dissociating togenerate positive charges and the group dissociating to generatenegative charges or the well-designed type C precursor can be used tocontrol such ratio in the modification.

In fact, at a pH at which electrostatic attraction is generated, morenet charges on the surface of the separation medium lead to a greateradsorption of the target substance. At a pH at which electrostaticrepulsion is generated, the adsorbed target substance can be efficientlyeluted as long as the net charges on the surface of the separationmedium and the net charge on the target substance are the same and havesufficient amounts. Theoretically, when the separation medium without anamphoteric dissociation covalently-modified layer on the surface is usedas a substrate for surface modification, the total molar amount of thegroup dissociating to generate negative charges and the groupdissociating to generate positive charges formed on the surface bycovalent modification is limited since there is a limitation in theamount or density of the reactive groups on the surface of the medium tobe modified. Different molar ratios of the group dissociating togenerate negative charges to the group dissociating to generate positivecharges may lead to difference in the pIm of the correspondingamphoteric dissociation separation mediums. For different targetsubstances, amphoteric dissociation ion exchange separation mediums withdifferent pIms are required to maximize the separation performance andmaximize the yield of target substances. Accordingly, less aliphaticcarboxyl group on the surface of the amphoteric dissociation ionexchange separation medium for adsorbing negatively charged targetsubstances is suitable to allow the medium for positive charge with alarger absolute value. Less aliphatic primary amine group or imidazolylgroup on the surface of the amphoteric dissociation ion exchangeseparation medium for adsorbing positively charged target substances issuitable to allow the medium for negative charge with a larger absolutevalue. Therefore, a wider range of the ratio of the total of thealiphatic primary amine group and the imidazolyl group to the aliphaticcarboxyl group results in a wider range of the pIm of the correspondingamphoteric dissociation ion exchange separation medium. It is requiredthat when used for separating negative ions, the amount of positivecharges on the surface of the amphoteric dissociation ion exchangeseparation medium is not lower than 90% of the maximum value at pH 3.0,and when used for separating positive ions, the amount of negativecharges on the surface of the amphoteric dissociation ion exchangeseparation medium is not lower than 90% of the maximum value at pH 11.0.

A general method for preparing the amphoteric dissociation ion exchangeseparation medium is to use a separation medium having a reactive groupbut no amphoteric dissociation modification layer on the surface as asubstrate to directly or indirectly but covalently link with anamphoteric dissociation group precursor having a corresponding group forcovalent linking to produce the desired amphoteric dissociationcovalently-modified layer. For the extraction of nucleic acids which arerequired to be amplified by PCR for detection, it is required that thenon-specific adsorption of the amphoteric dissociation ion exchangeseparation medium to small molecules is sufficiently low to reduce thecontent of interfering materials which are contained in the extractednucleic acids and can inhibit the PCR. Polyethylene glycol is a neutralhydrophilic polymer and is also a commonly-used modifier againstnon-specific protein adsorption. However, polyethylene glycol isactually a surfactant having an extremely high solubility in chloroformand a strong non-specific adsorption to small hydrophobic molecules.Therefore, the separation medium prepared by modification orpolymerization with a polyethylene glycol derivative is not suitable asa substrate for preparing the amphoteric dissociation ion exchangemedium of the invention, and the carboxyl group derivative and theprimary amino group derivative of the polyethylene glycol are also notsuitable as the amphoteric dissociation group precursor of theinvention. In particular, the polyethylene glycol molecules have astructure of linear straight chain so that the size exclusion betweenthe molecules is strong, which is not conducive to improving themodification degree of the amphoteric dissociation group, generatingdifficulty in increasing the separation capacity of the obtainedamphoteric dissociation separation medium and failing to reduce thenon-specific adsorption to small hydrophobic molecules by ensuring themodification degree. In this regard, it is not suitable for extractingnucleic acids and separating proteins with an ion exchange separationmedium bearing amphoteric dissociation groups on the surface whichprepared from direct polymerization of polyethylene glycol derivatives,or with an ion exchange separation medium bearing amphotericdissociation groups which is prepared by modification with thepolyethylene glycol derivatives. Therefore, the method of the inventionfor preparing an amphoteric dissociation ion exchange separation mediumby covalent modification is not suitable for the preparation of aseparation medium substrate with a linear macromolecule polymer butwithout an amphoteric dissociation group on the surface. A separationmedium prepared by polymerization of a linear macromolecule, even ifthere is an amphoteric dissociation group on the surface at pH 3.0-11.0,does not belong to the amphoteric dissociation ion exchange separationmedium of the invention.

Obviously, it is required that a small molecule as the amphotericdissociation group precursor and a linking reaction with a yield as highas possible at room temperature are employed to generate thehigh-density amphoteric dissociation modification layer to reduce thenon-specific adsorption to small hydrophobic molecules. In order toprepare the high-density modification layer to reduce the non-specificadsorption to hydrophobic substances, the amphoteric dissociation groupprecursor for modification is required to be controlled to have amolecular weight within 500 Daltons and have no hydrophobic bulkysubstituents. There is no requirement for anaerobic and anhydrousreaction to perform the amide formation and the substitution with athiol group so that they are suitable for the linking reactions requiredfor the modification. In the case that the amphoteric dissociationmodification layer is formed on the surface of the separation mediumwithout the amphoteric dissociation modification layer which is used asthe substrate, the aliphatic carboxyl group, the aliphatic primary andsecondary amine groups and the thiol reactive group on the surface ofthe separation medium substrate to be modified are suitable as reactivegroups, and the primary aliphatic amine group or the aliphatic thiolgroup is correspondingly required on the amphoteric dissociation groupprecursor as a group for the covalent linking. In general, when theseparation medium without an amphoteric dissociation covalently-modifiedlayer is used as the substrate, the reactive group contained on thesurface is one of the aliphatic carboxyl group, the aliphatic primaryamine group, the aliphatic secondary amine group and the thiol reactivegroup, where the thiol reactive group contains a small-size leavinggroup which is nucleophilically substituted by the thiol group easilyand leaves the surface of the separation medium. Such a small-sizeleaving group contains a halogen ion or a p-toluenesulfonate group. Inthe case that the reactive group is absent on the surface of theseparation medium without the amphoteric dissociationcovalently-modified layer, this separation medium can be provided withthe above reactive groups by derivatization to be used as the substratefor covalent modification.

The technology disclosed in Chinese Patent ZL201610963764.X uses azwitterion pair of which the surface consists of quaternary amine andsulfonic acid to prepare a separation medium with a hydrophilic surfaceby modification. By such technology was obtained a product FBD-MSP-FCOOHwhich is a micro magnetic bead. Besides the aliphatic carboxyl group asa functional group for coupling with other substances, the rest of thesurface of the FBD-MSP-FCOOH is a zwitterion pair modification groupwhich consists of quaternary amine and sulfonic acid and has a netcharge of zero at pH 2.0-13.0 due to the absence of amphotericdissociation, leading to a low non-specific adsorption of the surface tohydrophobic substances and proteins. Therefore, FBD-MSP-FCOOH is thedesired separation medium substrate without the amphoteric dissociationmodification layer for preparing the amphoteric dissociation ionexchange separation medium of the invention. Accordingly, the aliphaticcarboxyl group on the surface of this separation medium is activated (asshown in FIG. 2) and further derivatized to prepare a series ofamphoteric dissociation ion exchange micro magnetic beads with differentpIms (as shown in FIGS. 3 and 4). These ion exchange separation mediumscan adsorb nucleic acids at a weakly acidic pH based on theelectrostatic attraction of the surface positive charge to the targetsubstance, and the adsorbed nucleic acids can be efficiently released ata weakly basic pH based on electrostatic repulsion; or these ionexchange separation mediums can adsorb basic proteins at an appropriateneutral pH and a meta-alkalescent pH and the adsorbed proteins can bereleased under weakly acidic or strongly basic conditions, which is amore suitable ion exchange method for purifying the proteins of poorsolubility. This novel ion exchange separation medium provides higherselectivity to nucleic acids, and has a separation capacity exceeding 10times that of the Dynabeads Myone Silane (silanol magnetic beads,Thermo-Fisher Corporation), resulting in less interfering impurities inthe extracted nucleic acid. Therefore, when used as a PCR template, thenucleic acid extracted by such separation medium involves higher potencycompared to that extracted by the silanol magnetic beads. These micromagnetic beads are also suitable for the rapid purification of thePichia guilliermondii uricase which has a pK near 9.0 and has a poorsolubility at a neutral pH, and the elution yield is near 80% withreference to the enzymatic activity assay, to which the elution yield ofthe classical cation exchanger is far from satisfaction. However, whenthe amphoteric dissociation ion exchange separation medium is used toseparate proteins, the saturated adsorption of proteins may hinder theresponse of the dissociation state of the dissociation group on thesurface of the separation medium to the eluent pH, so that more eluentis required to elute the medium in batches to ensure the elutionefficacy.

In addition, the separation capacity is required to be accuratelycalibrated in the production process of the amphoteric dissociation ionexchange separation medium for controlling the product quality.Calibrating the separation capacity of these ion exchange separationmediums for nucleic acids as the target substance is the prerequisite toensure the reproducibility of test when the nucleic acids are used forhigh sensitivity analysis after extraction and concentration with suchmediums. In order to determine the property of the amphotericdissociation ion exchange separation medium of the invention, that is,its pIm, a simple method is required to determine variations of theseparation capacity and the elution efficacy for the charged targetsubstance with the adsorption pH. Quantitatively determining nucleicacids obtained by adsorption and separation with this product is themost direct method to calibrate the adsorption and separation capacityof this product. Fluorescence quantitative PCR, i.e., qPCR, is requiredfor the quantification of nucleic acids, where the minimum number ofcycles for the determination to reach the set signal is inverselyproportional to the logarithm of the template nucleic acid amount.However, this quantitative method involves low determination accuracy,high cost and time-consuming analysis, unsuitable for the qualitycontrol during the production process and inconvenient for thecharacterization of the properties, such as pIm, of the novel ionexchange separation medium of the invention. The spectrophotometricassay involves simple and rapid determination and good reproducibility,and measurement of the absorbance is easy to be standardized, suitablefor the calibration and tracing of the product quality and for thecharacterization of the amphoteric dissociation property of theamphoteric dissociation ion exchange separation medium of the invention.Though nucleic acids have strong ultraviolet absorbance, impuritiesintroduced in the adsorption and elution of nucleic acids may interferewith the measurement of ultraviolet absorbance. In addition, nucleicacids as the probe involve high cost and poor stability. A stablecharged colored small molecule organic material which is easy to bequantified by spectrophotometry can act as a color-developing probe,which is suitable for rapidly and easily calibrating the separationcapacity of the ion exchange separation medium and characterizing itspIm property. The application employs a water-soluble aromatic organicsubstance which has strong visible light absorbance and easydissociation as the color-developing probe, enabling efficientadsorption and elution of the probe at room temperature by adjusting pHand rapidly and accurately calibrating the adsorption and separationcapacity of the ion exchange separation medium based on the strongvisible light absorbance of the probe in the eluent. Moreover, the probecan be used for controlling the product quality and characterizing thepIm of the amphoteric dissociation ion exchange separation medium of theinvention (as shown in FIG. 1), thereby determining the specialamphoteric dissociation property and elution efficacy of the amphotericdissociation ion exchange separation medium.

The application further discloses a use method of the amphotericdissociation ion exchange separation medium, comprising: applying theamphoteric dissociation ion exchange separation medium to adsorb andseparate a target substance; wherein a common logarithm of adissociation constant obtained from the release of hydrogen ion from thetarget substance is pK or an isoelectric point of the target substanceis pIs; and the method further comprises:

a. selecting an amphoteric dissociation ion exchange separation medium;wherein the difference between the pIm of the amphoteric dissociationion exchange separation medium and the pK of the target substance is notless than 1.0;

b. adsorbing the target substance; wherein an environment pH is selectedto allow the type of net charge on the surface of the ion exchangeseparation medium to be opposite to the type of net charge of the targetsubstance, thereby resulting in adsorption of the target substance byelectrostatic attraction, wherein the environmental pH is locatedbetween the pK of the target substance and the pIm of the amphotericdissociation ion exchange separation medium while the difference betweenthe environmental pH and the pK of the target substance and thedifference between the environmental pH and the pIm of the amphotericdissociation ion exchange separation medium are both greater than 0.3;and

c. eluting the target substance; wherein when pH of an eluent is lowerthan the pIm of the amphoteric dissociation ion exchange separationmedium, the pH of the an eluent is at least 0.3 lower than the lower oneof the pK of the target substance and the pIm of the amphotericdissociation ion exchange separation medium; when the pH of the eluentis higher than the pIm of the amphoteric dissociation ion exchangeseparation medium, the pH of the an eluent is at least 0.3 higher thanthe higher one of the pK of the target substance and the pIm of theamphoteric dissociation ion exchange separation medium; in the use ofthe above eluent, the type of net charges on the surface of theamphoteric dissociation ion exchange separation medium is the same asthat on the target substance, thereby resulting in elution of the targetsubstance by electrostatic repulsion; and a water-soluble monovalentneutral inorganic salt is added to the eluent to promote the elution ofthe target substance.

The application also discloses a method for calibrating separationcapacity of the amphoteric dissociation ion exchange separation medium,comprising:

a) selecting an colored organic compound as a color-developing probe forthe calibration of the separation capacity of the amphotericdissociation ion exchange separation medium; wherein the colored organiccompound has a dissociation constant or an isoelectric point of pKa, amolecular weight less than 600 Daltons, a visible light absorptioncoefficient greater than 14 mM⁻¹·cm⁻¹, a solubility not less than 5.0μmol/L at pH 3.0-11.0, and a positive or negative net charge afterdissociation at pH 3.0-11.0;

b) calibrating the separation capacity of the amphoteric dissociationion exchange separation medium; wherein step b comprises:

b1) when the amphoteric dissociation ion exchange separation medium hasan isoelectric point pIm between 4.0 and 6.0, using a cationic probewith a dissociation constant pK or an isoelectric point pI at least 2.0greater than the pIm of the amphoteric dissociation ion exchangeseparation medium, or using an anionic probe with a dissociationconstant pK or an isoelectric point pI at least 2.0 lower than the pImof the amphoteric dissociation ion exchange separation medium; and

b2) when the amphoteric dissociation ion exchange separation medium hasthe pIm between 6.0 and 10.0, using the anionic probe with adissociation constant or an isoelectric point pKa at least 2.0 lowerthan the pIm of the amphoteric dissociation ion exchange separationmedium;

c) when calibrating the separation capacity of the amphotericdissociation ion exchange separation medium, selecting a buffer solutioncorresponding to an environmental pH to allow the amphotericdissociation ion exchange separation medium to adsorb a color-developingprobe by electrostatic attraction, wherein the environmental pH isbetween the dissociation constant pK or the isoelectric point pI of thecolor-developing probe and the pIm of the amphoteric dissociation ionexchange separation medium, and differs from the pIm of the amphotericdissociation ion exchange separation medium by not less than 1.3 anddiffers from the dissociation constant pK or the isoelectric point pI ofthe color-developing probe by not less than 0.5; selecting a buffersolution corresponding to an environmental pH to allow the type of netcharges on the surface of the amphoteric dissociation ion exchangeseparation medium to be the same as that of the color-developing probein the elution, thereby eluting a color-developing probe byelectrostatic repulsion, wherein the environmental pH is at least 1.3higher than the higher one or at least 1.3 lower than the lower one ofthe dissociation constant pK or the isoelectric point pI of thecolor-developing probe and the pIm of the amphoteric dissociation ionexchange separation medium; measuring the absorbance of thecolor-developing probe in an eluate followed by conversion into aseparation capacity for the color-developing probe; and

determining the effect of pH on the separation capacity of theamphoteric dissociation ion exchange separation medium for acolor-developing probe; wherein the minimum pH at which the separationcapacity for the color-developing probe with negative net charge reacheszero after the dissociation, or the maximum pH at which the separationcapacity for the color-developing probe with positive net charge reacheszero after the dissociation, is an approximation of the pIm of theamphoteric dissociation ion exchange separation medium.

That is, the variation of the separation capacity of the amphotericdissociation ion exchange separation medium for the color-developingprobe with pH is determined, where the minimum pH at which theseparation capacity for the color-developing probe dissociating togenerate negative ions reaches zero, or the maximum pH at which theseparation capacity for the color-developing probe dissociating togenerate positive ions reaches zero, is its pIm.

The amount of the adsorbed color-developing probe can be estimated bydetermining the amount of the remaining color-developing probe after theadsorption. Effect of the pH of the eluent on the elution efficacy canbe compared by determining the amount of the color-developing probe inthe eluate, thereby determining the efficacy of eluting the chargedsubstance based on the electrostatic repulsion of the invention.

There are fewer quaternary amine-type cationic color-developing probesbut many sulfonic acid-type anionic color-developing probes. Acid red 13as an anionic dye is a representative color-developing probe tocharacterize the separation capacity of the amphoteric dissociation ionexchange separation medium and determine the pIm.

Described below are the embodiments, and reagents and materials used inthe embodiments are listed as follows.

Silanol micro magnetic beads: Dynabeads Myone Silane (silanol magneticbeads) (Cat. no. 37002D), purchased from Thermo Fisher ScientificCorporation.

Magnetic separation rack: 8-hole magnetic separation rack, provided byBioCanal Scientific Inc. (Wuxi, Jiangsu).

Conventional PCR instrument: BioradT100 thermal cycler.

Fluorescence real-time quantitative PCR instrument: Biorad CFX Connect.

Carboxyl magnetic beads FBD-MSP-FCOOH: the carboxyl magnetic beadsFBD-MSP-FCOOH, prepared according to Chinese Patent ZL201610963764.X andneutral at pH 2.0-12.0 due to the modification layer of ion pairsconsisting of quaternary amine and sulfonic acid on the surface.

Pichia guilliermondii uricase RMGU expression plasmid: see Genbank forsequence with the Accession No. KY706244; isoelectric point 8.9; thefully synthetic coding sequence is ligated to an expression vector pDE1to obtain a tag-free expression plasmid which together with its proteinis called RMGU.

Acid red 13 stock solution: dissolved to 200 mM by distilled water,filtered by 0.22 μm microporous membrane, stored at 4° C. and dilutedbefore use.

Nucleic acid adsorption buffer: consisting of an acetic acid-sodiumacetate buffer (0.20 M) at pH 3.6-5.8, MES (20 mM) at pH 6.0-6.8 andHEPES (20 mM) at pH 7.0-8.0; unless otherwise specified, a nucleic acidadsorption buffer at pH 3.6 is employed.

Nucleic acid elution buffer: Tris-HCl (25 mM) at pH 8.0-9.0; unlessotherwise specified, a nucleic acid elution buffer at pH 8.9 isemployed.

Magnetic bead washing buffer: an acetic acid-sodium acetate buffer (0.20M) at pH 3.6.

Nucleic acid quantification: unless otherwise specified, thequantification is performed by measuring the absorbance at 260 nm usingNanodrop.

Nucleic acid electrophoresis: premixed ethidium bromide-agarose gelelectrophoresis recorded by a nucleic acid-protein imager with adetection limit of about 80 ng.

Uricase activity detection solution: consisting of borax (0.20M, pH 9.2)and uric acid (0.10 mM); consumption of 1.0 μmol of substrate per minuterefers to 1 unit.

Cell Lysis Solution 1: HEPES (20 mM, pH 7.6), for lysing recombinantRMGU-expressing E. coli cells.

Protein eluent 1: glycine-sodium hydroxide buffer (pH 10.0).

Protein electrophoresis: conventional SDS-PAGE; unless otherwisespecified, 5 g of the total protein for loading.

Protein concentration determination: using Bradford's dye binding methodor measuring the absorbance at 280 nm with Nanodrop.

The required chemical reagents are purchased from Tansoole and Aladdinreagent website with purity above 95%.

Described below are examples of the invention.

Example 1 Preparation of an Amphoteric Dissociation Ion ExchangeSeparation Medium with a Representative Structure

Step 1 Preparation of the following solutions (performing the filtrationwith 0.22 μm filtration membrane for simultaneous sterilization)

1) Lysine solution: prepared by adjusting a lysine solution (100 mM) topH 6.0 with hydrochloric acid (50 mM) and diluting the resultingsolution to 30 mM with water followed by filtration for use.

2) Histidine solution: prepared by adjusting a histidine solution (50mM) to pH 6.0 with hydrochloric acid (10 mM) and diluting the resultingsolution to 30 mM.

3) Glycine solution: prepared by adjusting a glycine solution (100 mM)to pH 6.0 with dilute sodium bicarbonate and diluting the resultingsolution to 30 mM with water followed by filtration for use.

4) N,N-dimethylethylenediamine solution: prepared by adjusting aN,N-dimethylethylenediamine solution (10 mM) to pH 6.0 with hydrochloricacid (0.10 M) and diluting the resulting solution to 10 mM.

5) Cysteine solution: prepared by adjusting an aqueous cysteine solution(50 mM) to pH 6.0 with sodium bicarbonate (10 mM) and diluting theresulting solution to 10 mM followed by filtration for use (required tobe used within 30 minutes after the preparation).

6) Mercaptoethylamine solution: prepared by adjusting an aqueousmercaptoethylamine solution (20 mM) to pH 6.0 with hydrochloric acid (10mM) and diluting the resulting solution to 10 mM (required to be usedwithin 30 minutes after the preparation).

7) Thioglycolic acid solution: prepared by adjusting an aqueousthioglycolic acid solution (20 mM) to pH 6.0 with sodium bicarbonate (10mM) and diluting the resulting solution to 10 mM (required to be usedwithin 30 minutes after the preparation).

8) 2-mercaptoimidazole solution: prepared by adjusting a2-mercaptoimidazole solution (20 mM) to pH 6.0 with sodium bicarbonate(10 mM) and diluting the resulting solution to 10 mM (required to beused within 30 minutes after the preparation).

Step 2 Activation of the carboxyl group of FBD-MSP-FCOOH into an activeester

FBD-MSP-FCOOH was repeatedly washed with a dry N,N-dimethylformamide(DMF), and then suspended with the dry DMF to produce a suspension of 10g/L. The suspension was added with dicyclohexylcarbodiimide (DCC) havinga weight 3 times that of the FBD-MSP-FCOOH and N-hydroxysuccinimide(NHS—OH) having a weight 1.5 times that of the FBD-MSP-FCOOH. Thereaction mixture was mixed uniformly and reacted continuously at 28-35°C. for 12 hours. The activated magnetic beads were washed three timeswith DMF to give an NHS active ester of the FBD-MSP-FCOOH magnetic bead,abbreviated as FBD-MSP-FCONHS (as shown in FIG. 2), where in each of thewashing, a mass-to-volume ratio of the FBD-MSP-FCOOH to the DMF is 1(g):100 (mL).

Step 3 Covalent modification of an active ester by formation of an amideto produce an amphoteric dissociation ion exchange separation medium (asshown in FIG. 3)

(1) FBD-MSP-FCONHS was suspended in a mixture prepared by the lysinesolution and the glycine solution in a volume ratio of 7:3 to produce amodification system for modification, where a mass-to-volume ratio ofthe FBD-MSP-FCONHS to the mixture is 1 (g):100 (mL). The modificationsystem was reacted under medium-speed stirring at room temperature for13 hours. The resulting magnetic beads were washed three times withdistilled water in a mass-to-volume ratio of 1 (g):100 (mL) to givecorresponding amphoteric dissociation ion exchange micro-magnetic beads,abbreviated as FBD-MSP-ZEWA.

(2) FBD-MSP-FCONHS was suspended in the lysine solution in amass-to-volume ratio of 1 (g):100 (mL) to produce a modification systemfor modification. The modification system was reacted under medium-speedstirring at room temperature for 13 hours. The resulting magnetic beadswere washed three times with distilled water in a mass-to-volume ratioof 1 (g):100 (mL) to give corresponding amphoteric dissociation ionexchange micro-magnetic beads, abbreviated as FBD-MSP-ZEWB.

(3) FBD-MSP-FCONHS was suspended in a mixture prepared by the lysinesolution and the N,N-dimethylethylenediamine solution in a volume ratioof 7:3 to produce a modification system for covalent modification, wherea mass-to-volume ratio of the FBD-MSP-FCONHS to the mixture is 1 (g):100(mL). The modification system was reacted under medium-speed stirring atroom temperature for 13 hours. The resulting magnetic beads were washedthree times with distilled water in a mass-to-volume ratio of 1 (g):100(mL) to give corresponding amphoteric dissociation ion exchangemicro-magnetic beads, abbreviated as FBD-MSP-ZEWC.

(4) FBD-MSP-FCONHS was suspended in the histidine solution in amass-to-volume ratio of 1 (g):100 (mL) to produce a modification systemfor covalent modification. The modification system was reacted undermedium-speed stirring at room temperature for 13 hours. The resultingmagnetic beads were washed three times with distilled water in amass-to-volume ratio of 1 (g):100 (mL) to give corresponding amphotericdissociation ion exchange micro-magnetic beads, abbreviated asFBD-MSP-ZEWD.

(5) FBD-MSP-FCONHS was suspended in the N,N-dimethylethylenediaminesolution in a mass-to-volume ratio of 1 (g):100 (mL) to produce amodification system for covalent modification. The modification systemwas reacted under medium-speed stirring at room temperature for 13hours. The resulting magnetic beads were washed three times withdistilled water in a mass-to-volume ratio of 1 (g):100 (mL) to givecorresponding amphoteric dissociation ion exchange micro-magnetic beads,abbreviated as FBD-MSP-ZEWE.

Step 4 Linking to a thiol reactive group for covalent modification toproduce an amphoteric dissociation ion exchange separation medium (asshown in FIG. 4)

(1) FBD-MSP-FCONHS was suspended in dimethylformamide in amass-to-volume ratio of 1 (g):100 (mL) to produce a suspension. Thesuspension was added with monochloroacetoethylenediamine (0.5 g) andreacted at room temperature under shaking for 4 hours to give aseparation medium to be modified which has a chloroacetyl group as athiol reactive group and is abbreviated as FBD-MSP-FCOCH₂Cl.

(2) FBD-MSP-FCOCH₂Cl was suspended in a mixture prepared by the cysteinesolution and the thioglycolic acid solution in a volume ratio of 7:3 toproduce a modification system for modification, where a mass-to-volumeratio of the FBD-MSP-FCOCH₂Cl to the mixture is 1 (g):100 (mL). Themodification system was reacted under medium-speed stirring at roomtemperature for 13 hours. The resulting magnetic beads were washed threetimes with distilled water in a mass-to-volume ratio of 1 (g):100 (mL)to give corresponding amphoteric dissociation ion exchangemicro-magnetic beads, abbreviated as FBD-MSP-ZEW1.

(3) FBD-MSP-FCOCH₂Cl was suspended in the cysteine solution in amass-to-volume ratio of 1 (g):100 (mL) to produce a modification systemfor covalent modification. The modification system was reacted undermedium-speed stirring at room temperature for 13 hours. The resultingmagnetic beads were washed three times with distilled water in amass-to-volume ratio of 1 (g):100 (mL) to give corresponding amphotericdissociation ion exchange micro-magnetic beads, abbreviated asFBD-MSP-ZEW2.

(4) FBD-MSP-FCOCH₂Cl was suspended in a mixture prepared by the cysteinesolution and the mercaptoethylamine solution in a volume ratio of 7:3 toproduce a modification system for covalent modification, where amass-to-volume ratio of the FBD-MSP-FCOCH₂Cl to the mixture is 1 (g):100(mL). The modification system was reacted under medium-speed stirring atroom temperature for 13 hours. The resulting magnetic beads were washedthree times with distilled water in a mass-to-volume ratio of 1 (g):100(mL) to give corresponding amphoteric dissociation ion exchangemicro-magnetic beads, abbreviated as FBD-MSP-ZEW3.

(5) FBD-MSP-FCOCH₂Cl was suspended in a mixture prepared by thethioglycolic acid solution and the 2-mercaptoimidazole solution in avolume ratio of 1:1 to produce a modification system for covalentmodification, where a mass-to-volume ratio of FBD-MSP-FCOCH₂Cl to themixture is 1 (g):100 (mL). The modification system was reacted undermedium-speed stirring at room temperature for 13 hours. The resultingmagnetic beads were washed three times with distilled water in amass-to-volume ratio of 1 (g):100 (mL) to give corresponding amphotericdissociation ion exchange micro-magnetic beads, abbreviated asFBD-MSP-ZEW4.

(6) FBD-MSP-FCOCH₂Cl was suspended in the mercaptoethylamine solution toproduce a modification system for covalent modification. Themodification system was reacted under medium-speed stirring at roomtemperature for 13 hours. The resulting magnetic beads were washed threetimes with distilled water in a mass-to-volume ratio of 1 (g):100 (mL)to give corresponding amphoteric dissociation ion exchangemicro-magnetic beads, abbreviated as FBD-MSP-ZEW5.

Certainly, the products prepared in the examples are merely illustrativeof how to prepare the amphoteric dissociation ion exchange separationmedium. The object of the invention can also be achieved by replacingthe material with one or more of the other above-described aliphaticcarboxyl groups dissociating to generate negative charge, primary,secondary and tertiary amine groups dissociating to generate positivecharge and imidazolyl groups dissociating to generate positive charge.

Example 2 Operation Process of Determination of the Adsorption andSeparation Capacity of the Amphoteric Dissociation Ion Exchange Mediumfor Acid Red 13

Step (1)

Effect of pH on the extinction coefficient of the acid red 13 wasmeasured and the results were shown in FIG. 5. No variation was observedin the extinction coefficient of the acid red 13 in a pH range from 4.0to 8.9, in which the extinction coefficient was maintained at 18.0(mM)⁻¹·cm⁻¹. The extinction coefficient of the acid red 13 describedbelow all used this value.

Step (2)

The amphoteric dissociation ion exchange separation medium was washedthree times with a buffer (pH 3.6) and then re-suspended with thisbuffer to produce a suspension.

Step (3) The acid red 13 was diluted with an adsorption buffer to notless than 5.0 mM.

Step (4)

The required amount of the ion exchange separation medium suspension wastransferred, subjected to the aqueous phase removal and resuspended in0.8 mL of the adsorption buffer to produce a resuspension. No more than50 μL of a dilute acid red 13 solution was added to the resuspension tothe desired final concentration. The reaction mixture was continuouslymildly mixed at room temperature by a four-dimensional mixer for 5-10minutes.

Step (5)

The acid red 13-binding magnetic beads were magnetically separated,washed twice with 0.80 mL of a washing buffer, resuspended in 0.80 mL ofan elution buffer and rotated and shaken by the four-dimensional mixerfor 5-10 minutes. The magnetic beads were removed and 0.70 mL of thesupernatant was used for measurement of the absorbance at 506 nm.

Step (6)

The maximum pH at which the separation capacity reaches zero wasapproximately determined according to the change of the separationcapacity of the amphoteric dissociation ion exchange separation mediumfor the acid red 13 with pH, and was used as the pIm of the testedamphoteric dissociation ion exchange separation medium.

Step (7)

The separation capacity for the acid red 13 in an adsorption buffer (pH3.6) indicated the separation capacity of the amphoteric dissociationion exchange separation medium, and was used to measure various magneticbeads with an amphoteric dissociation ion exchange group on the surface.

Example 3 Effect of Adsorption and Elution Conditions on the SeparationCapacity of the Amphoteric Dissociation Separation Medium (Referring toExample 2 for Operation)

Step (1)

In the case that the pH for adsorption was 3.6 and the concentration ofthe acid red 13 was 60 μM, the pH effect of the eluent was shown inTable 1.

The change of the pH of the eluent from 8.0 to 8.9 only had a strongeffect on the capacity of FDD-MSP-ZEWC and FBD-MSP-ZEW3 for separatingthe acid red 13, but the capacity did not increase significantly at thepH greater than 8.9. Unless otherwise specified, an eluent with pH 8.9was used for both the adsorbed acid red 13 and the adsorbed nucleicacid. The amount of the acid red 13 before the adsorption was used asthe total amount. Under the washing conditions used, the proportion ofthe acid red 13 in the washing solution, i.e., the washing loss rat, wasless than 20%. The reduction of acid red 13 in the supernatant indicatedthe total amount of adsorption. Except FBD-MSP-ZEWE and FBD-MSP-ZEW5,the elution rates of the acid red 13 adsorbed by the testedrepresentative amphoteric dissociation ion exchange magnetic beads at pH8.9 were greater than 95%. As for the FBD-MSP-ZEWE and the FBD-MSP-ZEW5,the elution rates were lower than 2% even at pH 8.9. The above resultssupported that the surface net charge can be reversed by adjusting thepH to promote the elution of charged substances.

TABLE 1 Effect of the pH of the eluent on the elution rate of acid red13 adsorbed by two kinds of amphoteric dissociation adsorptionseparation magnetic beads Amphoteric dissociation ion exchange Eluent pHmicro-magnetic Isoelectric 8.0 8.5 8.9 pH 8.9 Separation capacity μmol/gbead type point pIm Absorbance of eluent at 506 nm elution rate pH 3.6pH 4.5 FBD-MSP-ZEWA ~4.7 0.578 0.594 0.598 ~98% ~55 ~4 FBD-MSP-ZEWB ~6.70.832 0.845 0.849 ~97% 104 62 FBD-MSP-ZEWC ~7.7 0.701 0.760 0.786 ~95%102 74 FBD-MSP-ZEWD ~6.3 0.688 0.694 0.697 ~97%  82 42 FBD-MSP-ZEWE~9.4* 0.003 0.006 0.005  <1%  114*  98* FBD-MSP-ZEW1 ~4.3 0.556 0.5610.563 ~98%  54 ~3 FBD-MSP-ZEW2 ~6.1 0.812 0.830 0.833 ~97%  98 60FBD-MSP-ZEW3 ~6.8 0.656 0.678 0.682 ~95% 108 71 FBD-MSP-ZEW4 ~5.8 0.6240.634 0.636 ~97%  76 39 FBD-MSP-ZEW5 ~9.2* 0.003 0.005 0.006  <3%  102* 96* *indicated an extremely low elution efficacy at pH 8.9; theseparation capacity was estimated based on the reduction of the acid red13 in the supernatant after the adsorption and then used to estimate itspIm.

Step (2)

In the case that the adsorption and the elution were respectivelyperformed at pH 3.6 and 8.9 and the final concentration of acid red 13in the adsorption buffer was 60 μM, the relationship between theadsorption separation amount and the effect was determined. The resultsshowed that the amount of acid red 13 obtained in the eluate afterseparation was in linear response to the amount of FBD-MSP-ZEWB within160 μg (as shown in FIG. 6). Moreover, when different concentrations ofacid red 13 were used in the adsorption reaction system in the case of0.10 mg of FBD-MSP-ZEWA, the amount of acid red 13 in the eluate was innearly linear response to the amount of acid red 13 in the adsorptionreaction system within a limited range (as shown in FIG. 7). Suchresults indicated that using acid red 13 as a color-developing probe wassuitable for the method for calibrating the separation capacity of theseamphoteric dissociation separation mediums. In addition, the resultsalso supported that the amphoteric dissociation ion exchanger designedby the invention was suitable for efficiently recycling and reusing thewater-soluble dye in the dyeing and weaving waste liquid, therebyfacilitating the reuse and reducing environmental pollution.

Step (3)

pH of the adsorption reaction also showed significant effect on theseparation capacity of the amphoteric dissociation ion exchangeseparation medium (Table 1), and the representative data was shown inFIG. 8. The separation capacity of the magnetic beads for acid red 13was reduced at pH of greater than 4.0, indicating the continuousreduction in the positive charge. The pH of the adsorption reactionsystem should be located between 3.6-4.0 to achieve the maximumseparation capacity. The minimum pH at which the separation capacity foracid red 13 was near zero was the pIm, and the pIms of the threeexemplary magnetic beads were significantly different (as shown in FIG.8). At pH 8.0, the adsorption capacities of the three magnetic beads inFIG. 8 for acid red 13 were close to zero, indicating that the netcharge on the surface of the three beads was negative. Based on this,the pIm of the prepared amphoteric dissociation ion exchange separationmedium can be estimated. In addition, the difference in the pIms alsodemonstrated the pH effect of the eluent and further supported thedesign concept of the invention and the application of the resulting ionexchanger (Table 1).

Step (4)

The FBD-MSP-ZEWA (0.10 mg), acid red 13 (60 μM), an adsorption system(0.80 mL, pH 3.6) and an eluent (0.8 mL in total, pH 8.9) were used, andthe FBD-MSP-ZEWA was independently diluted. The separation capacity foracid red 13 was repeatedly determined to be (55±2) μmol/g magnetic bead(n=5), which indicated the high precision of this method, suitable forcalibrating the separation capacity of the amphoteric dissociation ionexchange separation medium of the invention. The magnetic beads weremeasured based on the separation amount for acid red 13 at pH 3.6 in thefollowing examples.

Example 4 Application of FBD-MSP-ZEWB and FBD-MSP-ZEWA in the Extractionof Plasmid DNA

Step (1)

The RMGU plasmid was used as a model. After E. coli cells underwentscale-up culture, the plasmid was extracted as a nucleic acid samplebased on the combination of the base-lysed E. coli cells and the spincolumn using a spin-column plasmid extraction kit (Tiangen).

Step (2)

Unless otherwise specified, the binding of magnetic beads to a plasmidwas all performed at pH 3.6 and the elution was performed at pH 8.9.

Step (3)

Silanol magnetic beads (0.40 mg, Thermo-Fisher Corporation),FBD-MSP-ZEWA (5.5 nmol, 0.10 mg), an adsorption system (0.20 mL intotal) and an eluent (40 μL) were used and nanodrop was employed todetermine the amount of nucleic acid through the measurement of A260.The response curve of the nucleic acid amount obtained from theseparation to the excess nucleic acid amount in the adsorption reactionsystem was shown in FIG. 9. It can be seen that in the case of excessnucleic acid, the capacity of the FBD-MSP-ZEWA for separating nucleicacids was significantly higher than 4 times that of the silanol magneticbead (Thermo-Fisher Corporation). The maximum amount of nucleic acidadsorbed by the magnetic beads is obtained by subtracting the amount ofnucleic acid remained in the supernatant after adsorption from the totalamount of nucleic acid in the adsorption reaction system, and then usedfor calculating the elution effect. At pH 8.9, the elution effect of thenucleic acid adsorbed by FBD-MSP-ZEWA was greater than 85% while that ofthe nucleic acid adsorbed by the silanol magnetic bead (Thermo-FisherCorporation) was lower than 30%, which supported the design concept ofthe invention.

Step (4)

An adsorption system was 0.20 mL in total and contained 5.0 μg of aplasmid, and the eluent was 40 μL. A response curve of the obtainedamount of nucleic acid to the amount of magnetic beads was shown in FIG.10. It can be seen that the potency of FBD-MSP-ZEWB for separatingnucleic acid may exceed 9 times that of the silanol magnetic bead(Thermo-Fisher Corporation). The separation capacity of FBD-MSP-ZEWB fornucleic acid was significantly higher than that of FBD-MSP-ZEWA, and thedifference was consistent with the difference in the capacity of the twofor separating acid red 13. Such difference also supported the designconcept of the invention and the practicality of the designed ionexchanger.

Step (5)

An adsorption system was 0.20 mL in total and contained 0.40 μg of aplasmid. The adsorption system was separated by FBD-MSP-ZEWB (50 nmol,˜0.15 mg), silanol magnetic bead (1.0 mg, Thermo-Fisher Corporation) andsilanol magnetic bead in a Biomiga kit (1.0 mg), respectively. Theeluent was 40 μL. 2.5 μL of the nucleic acid extract was transferred toa PCR reaction system (50 μL). The PCR products obtained by differentcycles or amplification times were detected by agarose electrophoresis,and the results were shown in FIG. 11, where M indicated the referenceof the nucleic acid molecular weight; 1, 4, 7, 10, 13, 16: plasmidsextracted by 0.15 mg of FBD-MSP-ZEWB; 2, 5, 8, 11, 14, 17: plasmidsextracted by 1.0 mg of the silanol magnetic bead (Thermo-FisherCorporation); 3, 6, 9, 12, 15, 18: plasmids extracted by 1.0 mg of theBiomiga silanol magnetic bead (the source of the silanol magnetic beadsin the kit was unknown); 1, 2, 3: 10 PCR cycles in total; 4, 5, 6: 13PCR cycles in total; 7, 8, 9: 16 PCR cycles in total; 10, 11, 12: 19 PCRcycles in total; 13, 14, 15: 22 PCR cycles in total; and 16, 17, 18: 25PCR cycles in total.

As a template, the nucleic acid extracted by the FBD-MSP-ZEWB (50 nmol)showed significantly higher potency than that respectively extracted by1.0 mg of the silanol magnetic bead (Thermo-Fisher Corporation) and 1.0mg of the Biomiga silanol magnetic bead (the source of the silanolmagnetic beads in the kit was unknown). The FBD-MSP-ZEWB was moresuitable for extracting nucleic acids for PCR, supporting theapplication of the ion exchanger.

Example 5 Application of FBD-MSP-ZEWB in the Purification of TraceRecombinant RMGU-Expressing Protein by Ion Exchange

MGU had a strong adsorption to carboxymethyl or phosphorylated celluloseand Toyopear sulfonic acid hydrophilic macroporous resin cation exchangemedium, so that the adsorbed MGU cannot be eluted even using an eluentwith a volume 10 times that of the gel (Protein J (2016) 35: 318-329, asa reference cited herein). The FBD-MSP-ZEWB, with a pIm close to 6.6 anddiffering from the pIs of RMGU by more than 2.0 pH units, was selectedto improve the separation capacity of the amphoteric dissociation ionexchanger and ensure the stability of RMGU, so that a buffer solutionwith a pH between the pIm and the pIs and close to the neutrality wasselected to lyse the cells and the lysed cells can be directly used foradsorption and separation after filtration with 0.22 μm microporousmembrane.

Step (1)

The cells were induced to express RMGU, lysed in the cell lysis solution1 under ultrasonication and centrifuged at 12,000 rpm. The supernatantwas filtered to give a recombinant RMGU-expressing crude enzyme sample,which had a protein content of 3.25 g/L, an activity of 3.27 kU/L and aspecific activity of 1.01 kU/g.

Step (2)

The FBD-MSP-ZEWB magnetic bead (3.0 μmol) was washed three times withthe cell lysis solution, added with different amounts of the crudeenzyme samples and mixed at room temperature for 10 minutes to produce asuspension. The suspension was separated magnetically. The resultingmagnetic beads were washed with the cell lysis solution 1, added withthe protein eluent 1 and mixed at room temperature for 30 minutes togive an eluate. After collecting the eluate, the magnetic beads wereadded with the same amount of the protein eluent 1 and re-eluted for 30minutes. The elution was repeated several times. The concentration ofthe protein was determined by measuring the absorbance at 280 nm usingNanodrop, and the results were shown in Table 2. It can be seen thatRMGU can be efficiently purified by FBD-MSP-ZEWB, and the specificactivity of the resulting enzyme exceeded that of the enzyme purified byDEAE-cellulose adsorption three times (Protein J (2016)35:318-329). Thehighest elution efficacy of the adsorbed RMGU was nearly 80% whencalculated according to the activity, while the MGU adsorbed by theclassical cation exchange medium fails to be eluted. Moreover, theresults of the batchwise elution with an eluent indicated that the RMGUwith the highest activity and concentration was obtained by the secondelution. Therefore, 80 μL of each of the second eluates was lyophilized,directly dissolved with a SDS-PAGE loading buffer (25 μL) under heatingand analyzed by SDS-PAGE at the same loading amount of protein. Theresults showed that the purity of RMGU purified by FBD-MSP-ZEWB ionexchange once was very high and significantly higher than that of RMGUpurified by DEAE-cellulose three times (FIG. 12; By comparison withProtein J (2016)35:318-329).

TABLE 2 Rapid separation of RMGU with FBD-MSP-ZEWB (3.0 μmol in total)(ND indicated that the protein concentration was too low to detect)Sample amount 0.20 mL Crude enzyme 1.00 mL Crude enzyme 4.00 mL Crudeenzyme Protein Protein Protein Activity concentration Activityconcentration Activity concentration Index (kU/L) (g/L) (kU/L) (g/L)(kU/L) (g/L) Supernatant after 2.41 2.72 2.38 3.14 2.76 3.20 adsorptionAdsorbed amount 0.086 53 0.44 55 1.02 50 (Expressed by activity/U orprotein/μg) The first eluate 0.09 ND 0.20 ND 0.23 ND (0.20 mL) Thesecond eluate 0.32 0.028 0.57 0.053 0.71 0.09 (0.10 mL) The third eluate0.08 ND 0.13 ND 0.13 ND (0.20 mL) Total activity of 0.068 0.13 0.15 theeluates (U) Recovery rate of 79 30 15 the activity after the elution (%)The highest ~11.4 10.8 7.9 specific activity of the eluate (kU/g)Purification fold ~12.2 10.6 7.8

Example 6 Extraction of RMGU Plasmid with FBD-MSP-ZEWB for QuantitativePCR

Step (1)

E. coli cells were transformed with RMGU and then subjected to scale-upculture. The cells were collected, lysed with a base and extracted by aspin column method for a plasmid as a preliminarily-purified plasmid.The preliminarily-purified plasmid was adjusted to pH 3.6 with aceticacid (0.20 M) for subsequent purification.

Step (2)

The plasmid obtained in step (1) was further purified with FBD-MSP-ZEWB.The eluate was adjusted to pH 3.6 with acetic acid (0.20 M) and purifiedwith FBD-MSP-ZEWB to give a plasmid standard, which was diluted to theappropriate concentration as described in Step (6).

Step (3)

FBD-MSP-ZEWB (50 nmol), Dynabeads Myone Silane (1.0 mg) and Biomiga kitmagnetic beads (1.0 mg) were directly eluted with 40 μL of an eluent (25mM Tris-HCl, pH 8.9), respectively, to obtain impurities from each ofthe magnetic beads.

Step (4)

The impurities from each of the magnetic beads were added with the sameamount of the plasmid standard and used as the magnetic bead blank forfluorescence real-time quantitative PCR (qPCR) to compare the content ofimpurities, which can inhibit the qPCR, in the nucleic acids extractedby different magnetic beads.

Step (5)

The plasmid purified by the spin column was diluted. The diluted plasmid(20 μL, 0.40 μg) was adjusted to pH 3.6 with sodium acetate (45 μL,0.20M) and acetic acid (235 μL, 0.20 M), respectively extracted withFBD-MSP-ZEWB (50 nmol, 0.15 mg), Dynabeads myone Silane (1.0 mg) andBiomiga kit magnetic beads (1.0 mg) and eluted with Tris-HCl buffer (40μL, 25 mM, pH 8.9) to give test solutions of corresponding magneticbead-extracted nucleic acid.

Step (6)

Samples for qPCR were listed as follows.

1—the plasmid standard

The RMGU plasmid was purified once by the spin column method and twotimes with FBD-MSP-ZEWB and determined to have a plasmid concentrationof 26.9 mg/L by measuring the absorbance at 260 nm. The purified plasmidwas diluted 1125 times with the eluent and used as an application liquidof the plasmid standard. The application liquid of the plasmid standardwas diluted 4, 16, 64, 256 and 1024 times for qPCR, respectively.

2—FBD-MSP-ZEWB blank: prepared by an individual magnetic bead eluate (20μL, 50 nmol) and a 64-fold diluted application liquid of the plasmidstandard (20 μL).

3—Dynabeads Myone Silane blank: prepared by an individual magnetic beadeluate (20 μL, 1.0 mg) and a 64-fold diluted application liquid of theplasmid standard (20 μL).

4—Biomiga magnetic bead blank: prepared by an individual magnetic beadeluate (20 μL, 1.0 mg) and a 64-fold diluted application liquid of theplasmid standard (20 μL).

5—FBD-MSP-ZEWB test solution

0.40 g of the plasmid was extracted with 50 nmol of FBD-MSP-ZEWBmagnetic beads and diluted 360 times with the eluent to give theFBD-MSP-ZEWB test liquid.

6—Dynabeads Myone Silane test solution

0.40 g of the plasmid was extracted with 1.0 g of Dynabeads Myone Silaneand diluted 360 times with the eluent to give the Dynabeads Myone Silanetest liquid.

7—Biomiga magnetic bead test solution

0.40 g of the plasmid was extracted with 1.0 g of Biomiga magnetic beadand diluted 360 times with the eluent to give the Biomiga magnetic beadtest solution.

Step (7)

Fluorescence real-time quantitative PCR was performed as follows. BioradCFX96 was used according to the standard procedure; the fluorescent dyewas Sybrgreen; and the PCR reaction system (25 μL in total) contained anenzyme, a trinucleotide, a primer for controlling the product to be 150bp, Sybrlgreen and a template (2.5 μL). The process of tracing themagnetic bead blanks was shown in FIG. 13. The process of tracing themagnetic bead test solutions was shown in FIG. 14 and the response curvewas shown in FIG. 15. The plasmid concentration in the applicationliquid of the plasmid standard was set to 24 pg/L, and the gradient ofthe total amount of the plasmid in each reaction system of the responsecurve was sequentially 60 pg, 15 pg, 3.75 pg, 0.94 pg, 0.24 pg and 0.06pg, of which the natural logarithms were set as the abscissa to plot anegative response curve (see FIG. 15 for details).

Step (8)

It can be seen from Table 3 that when used for extracting nucleic acids,the impurities in the FBD-MSP-ZEWB showed the least inhibitory effect onPCR compared to those in the two silanol magnetic beads, and moreinhibitory impurities were observed in the Biomiga magnetic beads. For aplasmid with a trace amount, given the amount of magnetic beads (mass)and the amount of a template for qPCR determination, the potency of thenucleic acid extracted by FBD-MSP-ZEWB for qPCR exceeded 10 times thatof the nucleic acid extracted by Dynabeads Myone Silane for qPCR. TheBiomiga magnetic bead treatment resulted in the lowest template amountafter qPCR due to the obviously more impurities inhibiting qPCR.

TABLE 3 Determination of the amount of plasmid extracted by magneticbeads and interfering impurities in various magnetic bead extracts byfluorescence real-time quantitative PCR Biomiga Dynabeads BiomigaDynabeads magnetic Myone Silane FBD-MSP-ZEWB magnetic Myone SilaneFBD-MSP-ZEWB bead test Samples Blank Blank bead blank test solution testsolution solution Cq 25.56 24.45 25.74 20.46 18.60 21.01 Plasmid 0.430.94 0.35 14.09 51.88 9.58 amount (pg) Ratio to 1.0 2.2 0.81 1.0 3.70.68 Dyna

The above examples demonstrated the practicality of the amphotericdissociation ion exchange separation medium of the invention. The mediahad strong adsorption and elution properties for acid red 13 dyes, sothat acid red 13 can be used for characterizing the ion exchangeseparation medium and the separation medium can also be used to recycleand reuse the acid red water-soluble dyes in printing and dyeing wasteliquid. The elution and measurement which were performed based on thebinding of the ion exchange separation medium to acid red 13 canfacilitate the characterization of the pIm of the prepared amphotericdissociation ion exchange separation medium and pH effect of the eluent.The amphoteric dissociation ion exchange separation medium with anappropriate pIm can be used to efficiently and rapidly purify theprotein and efficiently extract nucleic acids. Moreover, the extractednucleic acids were more suitable for PCR. The results of thefluorescence real-time quantitative PCR indicated that based on the massof the micro-magnetic beads, the potency of the nucleic acid extractedby the amphoteric dissociation ion exchange separation medium for PCRwas near 10 times that of the nucleic acid extracted by theThermo-Fisher Dynabeads Myone Silane silanol magnetic beads.

Finally, the above examples are merely illustrative of the technicalsolutions of the invention and are not intended to limit the scope ofthe invention. It should be understood that modifications and equivalentsubstitutions made by those skilled in the art without departing fromthe spirit and scope of the invention should fall within the scope asdefined by the appended claims.

We claim:
 1. An amphoteric dissociation ion exchange separation medium,wherein a surface of the amphoteric dissociation ion exchange separationmedium is an amphoteric dissociation covalently-modified layer; theamphoteric dissociation covalently-modified layer has an isoelectricpoint (pIm) that is an environmental pH value at which a net charge onthe surface of the amphoteric dissociation ion exchange separationmedium is zero; wherein when the environmental pH value is lower thanthe pIm, the net charge on a surface of the amphoteric dissociationcovalently-modified layer is positive and the amphoteric dissociationion exchange separation medium acts as an anion exchanger; when theenvironmental pH value is higher than the pIm, the net charge on thesurface of the amphoteric dissociation covalently-modified layer isnegative and the amphoteric dissociation ion exchange separation mediumacts as a cation exchanger; the amphoteric dissociationcovalently-modified layer on the surface of the amphoteric dissociationion exchange separation medium comprises both a group which dissociatesto generate positive charges only, and a group which dissociates togenerate negative charges only; wherein the group which dissociates togenerate negative charges only is an aliphatic carboxyl group, and thegroup which dissociates to generate positive charges only is one or moregroups selected from the group consisting of an aliphatic primary aminegroup, an aliphatic secondary amine group, an aliphatic tertiary aminegroup and an imidazolyl group; the amphoteric dissociationcovalently-modified layer is derived from covalent modification of asurface of a separation medium substrate with an amphoteric dissociationgroup precursor; the surface of the separation medium substrate does notcontain a long linear chain having a length of more than 9 carbon atoms,but contains a reactive group for covalently linking with the amphotericdissociation group precursor to form the amphoteric dissociationcovalently-modified layer; the reactive group is any one group selectedfrom the group consisting of an aliphatic primary amine group, analiphatic secondary amine group, an aliphatic carboxyl group and a thiolreactive group; wherein the thiol reactive group contains a small-sizegroup that is substituted by a thiol to leave from the surface of theseparation medium substrate; the amphoteric dissociation group precursoris a hydrophilic material having a molecular weight less than 500Daltons and no hydrocarbon chains or hydrocarbon rings with more than 5successive carbon atoms; wherein the amphoteric dissociation groupprecursor contains an alkylthiol group or an aliphatic primary aminegroup as a covalently-linking group and an aliphatic carboxyl group asthe group which dissociates to generate negative charges only, and/orone or more groups selected from the group consisting of an aliphaticprimary amine group, an aliphatic secondary amine group, an aliphatictertiary amine group and an imidazolyl group as a group whichdissociates to generate positive charges only; the amphotericdissociation group precursor comprises a type A precursor, a type Bprecursor and a type C precursor, wherein the type A precursor is anamphoteric dissociation group precursor comprising only the group whichdissociates to generate negative charge besides the covalently-linkinggroup, the Type B precursor is an amphoteric dissociation groupprecursor comprising only the group which dissociates to generatepositive charge besides the covalently-linking group, and the type Cprecursor is an amphoteric dissociation group precursor comprising thegroup which dissociates to generate negative charge and the group whichdissociates to generate positive charge besides the covalently-linkinggroup; wherein the type C precursor is selected from the groupconsisting of lysine, ornithine, histidine,N,N-dicarboxymethylethylenediamine, cysteine, 3-thiolhistidine, 3-thiollysine and 3-thiol glutamic acid; the type A precursor is selected fromthe group consisting of glutamic acid, 3-thiol-1,5-glutaric acid,thioglycolic acid and tris-(carboxymethyl)-aminomethane; and the type Bprecursor is selected from the group consisting of mercaptoethylamine,3-thiol-2-hydroxypropylamine, 2-mercaptoimidazole, diethylenetriamine,N,N-dimethylaminoethylenediamine and tetra-(aminomethyl)-methane.
 2. Theamphoteric dissociation ion exchange separation medium of claim 1,wherein when the environmental pH value is lower than the pIm, thenumber of positive charges on the surface of the amphoteric dissociationcovalently-modified layer increases as the difference between the pImand the environmental pH value increases; when the environmental pHvalue is higher than the pIm, the number of negative charges on thesurface of the amphoteric dissociation covalently-modified layerincreases as the difference between the pIm and the environmental pHvalue increases; and the environmental pH value is within a pH rangetolerated by both a target substance and the amphoteric dissociation ionexchange separation medium.
 3. The amphoteric dissociation ion exchangeseparation medium of claim 1, wherein the separation medium substrate iscovalently modified with the amphoteric dissociation group precursor byone of the following methods: a) using one or more type C precursors;wherein, when two or more type C precursors are used in combination, thetype C precursors are mixed in a given ratio for use, and the precursorsfor mixing contain the same covalently-linking group; b) mixing one ormore type A precursors with one or more type B precursors in a givenratio for use; wherein the precursors for mixing contain the samecovalently-linking group; c) mixing only one or more type A precursorswith one or more type C precursors in a given ratio; wherein theseprecursors for mixing contain the same covalently-linking group; and d)mixing only one or more type B precursors with one or more type Cprecursors in a given ratio; wherein the precursors for mixing containthe same covalently-linking group.
 4. The amphoteric dissociation ionexchange separation medium of claim 3, wherein when the amphotericdissociation group precursor is used to covalently modify the separationmedium substrate, a molar ratio of the total amount of aliphaticprimary, secondary, tertiary amine groups and imidazolyl groups to thetotal amount of aliphatic carboxyl groups in the amphoteric dissociationgroup precursor is limited to 1:6 to 6:1.
 5. The amphoteric dissociationion exchange separation medium of claim 1, wherein when an aliphaticsecondary amine and/or an aliphatic tertiary amine are/is used in anamphoteric dissociation group precursor as the group which dissociatesto generate positive charge, the total molar amount of the aliphaticsecondary amine and the aliphatic tertiary amine does not exceed 30% ofthe total molar amount of the aliphatic primary amine and the imidazolylgroup in the precursors used.
 6. The amphoteric dissociation ionexchange separation medium of claim 4, wherein the number of positivecharge on the surface of the amphoteric dissociation ion exchangeseparation medium is not less than 90% of the maximum value at pH 3, andthe number of negative charge on the surface of the amphotericdissociation ion exchange separation medium is not less than 90% of themaximum value at pH
 11. 7. A use method of the amphoteric dissociationion exchange separation medium of claim 1, comprising: applying theamphoteric dissociation ion exchange separation medium to separate atarget substance; wherein a common logarithm of a dissociation constantobtained from the release of hydrogen ion from the target substance ispK or an isoelectric point of the target substance is pI; wherein themethod further comprises: a. selecting an amphoteric dissociation ionexchange separation medium; wherein difference between the pIm of theamphoteric dissociation ion exchange separation medium and the pK or pIof the target substance is not less than 1.0; b. adsorbing the targetsubstance; wherein an environment pH is selected to allow the type ofnet charges on the surface of the ion exchange separation medium to beopposite to the type of net charges of the target substance, therebyresulting in adsorption of the target substance by electrostaticattraction, wherein the environmental pH is between the pK or pI of thetarget substance and the pIm of the amphoteric dissociation ion exchangeseparation medium and the difference between the environmental pH andthe pK or pI of the target substance and the difference between theenvironmental pH and the pIm of the amphoteric dissociation ion exchangeseparation medium are both greater than 0.3; and c. eluting the targetsubstance; wherein when pH of an eluent is lower than the pIm of theamphoteric dissociation ion exchange separation medium, the pH of theeluent is at least 0.3 lower than the lower one of the pK or pI of thetarget substance and the pIm of the amphoteric dissociation ion exchangeseparation medium; when the pH of the eluent is higher than the pIm ofthe amphoteric dissociation ion exchange separation medium, the pH ofthe eluent is at least 0.3 higher than the higher one of the pK or pI ofthe target substance and the pIm of the amphoteric dissociation ionexchange separation medium; in the use of the above eluent, the type ofnet charges of the surface of the amphoteric dissociation ion exchangeseparation medium is the same as that of the target substance, therebyresulting in elution of the target substance by electrostatic repulsion;and a water-soluble monovalent neutral inorganic salt is added to theeluent to promote the elution of the target substance.
 8. A method forcalibrating separation capacity of the amphoteric dissociation ionexchange separation medium of claim 1, comprising: a) selecting ancolored organic compound as a color-developing probe for the calibrationof the separation capacity of the amphoteric dissociation ion exchangeseparation medium; wherein the colored organic compound has adissociation constant of pK or an isoelectric point of pI, a molecularweight less than 600 Daltons, a visible light absorption coefficientgreater than 14 mM⁻¹·cm⁻¹, a solubility not less than 5.0 μmol/L at pH3.0-11.0, and a positive or negative net charge after dissociation at pH3.0-11.0; b) calibrating the separation capacity of the amphotericdissociation ion exchange separation medium; wherein step b comprises:b1) when the amphoteric dissociation ion exchange separation medium hasan isoelectric point pIm between 4.0 and 6.0, using a cationic probewith a dissociation constant of pK or an isoelectric point pI at least2.0 greater than the pIm of the amphoteric dissociation ion exchangeseparation medium, or using an anionic probe with a dissociationconstant pK or an isoelectric point pI at least 2.0 lower than the pImof the amphoteric dissociation ion exchange separation medium; and b2)when the amphoteric dissociation ion exchange separation medium has thepIm between 6.0 and 10.0, using the anionic probe with a dissociationconstant of pK or an isoelectric point pI at least 2.0 lower than thepIm of the amphoteric dissociation ion exchange separation medium; c)when calibrating the separation capacity of the amphoteric dissociationion exchange separation medium, selecting a buffer solutioncorresponding to an environmental pH to allow the amphotericdissociation ion exchange separation medium to adsorb a color-developingprobe by electrostatic attraction, wherein the environmental pH isbetween the dissociation constant pK or the isoelectric point pI of thecolor-developing probe and the pIm of the amphoteric dissociation ionexchange separation medium, and differs from the pIm of the amphotericdissociation ion exchange separation medium by not less than 1.3 anddiffers from the dissociation constant pK or the isoelectric point pI ofthe color-developing probe by not less than 0.5; selecting a buffersolution corresponding to an environmental pH to allow the type of netcharges on the surface of the amphoteric dissociation ion exchangeseparation medium to be the same as that of the color-developing probein the elution, thereby eluting a color-developing probe byelectrostatic repulsion, wherein the environmental pH is at least 1.3higher than the higher one or at least 1.3 lower than the lower one ofthe dissociation constant pK or the isoelectric point pI of thecolor-developing probe and the pIm of the amphoteric dissociation ionexchange separation medium; measuring the absorbance of thecolor-developing probe in an eluate followed by conversion into aseparation capacity to the color-developing probe; and determining pHeffect on the separation capacity of the amphoteric dissociation ionexchange separation medium to a color-developing probe; wherein theminimum pH at which the separation capacity to the color-developingprobe with negative net charge reaches zero, or the maximum pH at whichthe separation capacity to the color-developing probe with positive netcharge reaches zero, is an approximation of the pIm of the amphotericdissociation ion exchange separation medium.